Recombinant:Article Title: SEPHS2 loss reprograms cancer metabolism from oxidative phosphorylation to gluconeogenesis via PCK1 stabilization.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies SEPHS2 proteintech Cat#14109-1-AP; Lot#00124159; RRID: AB_2878014 LA/LP Polyclonal antibody(SEPSECS) proteintech Cat#11551-1-AP; Lot#00121618; RRID:AB_2186853 PCK1 (D12F5) Rabbit mAb Cell Signaling Technology Cat#12940S; Lot#3; RRID:AB_2687968 Anti FLAG Nanobody Agarose Beads ALPALIFEBIO Cat#KTSM1308; Lot#240810; RRID:AB_3740166 Anti-HA tag Rabbit mAb PTM bio Cat#PTM-5389; Lot#RL110916; RRID:AB_3740293 Anti-DYKDDDDK Tag Mouse mAb PTM bio Cat#PTM-6075; Lot#MN120818; RRID:AB_3740294 Pan-Acetyl-Lysine Mab Abmart Cat#M30069S; Lot#334095; RRID: AB_2936273 β-actin Beta Actin Monoclonal antibody proteintech Cat#66009-1-Ig; Lot#10052137; RRID: AB_2687938 HRP-conjugated GAPDH Monoclonal antibody proteintech Cat#HRP-60004; Lot#21020050; RRID: AB_2107436 HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) proteintech Cat#SA00001-2; Lot#20001102; RRID: AB_2722564 HRP-conjugated Affinipure Goat Anti-Mouse IgG(H + L) proteintech Cat#SA00001-1; Lot#20001336; RRID: AB_2722565 Ki-67 Invitrogen Cat#MA5-14520; Lot#ZC4251801; RRID: AB_10979488 BrdU HUA BIO Cat#HA721283; Lot#H651616016; RRID: AB_3072400 Chemicals, peptides, and recombinant proteins DMEM VivaCell Cat#C3113-0500; Lot#2523153 Minimum Essential Medium Eagle,(MEM-E) VivaCell Cat#C3050-0500; Lot#2421230 glucose-free DMEM Wisent Cat#319-061-CL; Lot#319061013 13C6-glucose Cambridge Isotope Laboratories Cat#CLM-1396-1; Lot#PR-34213 Fetal bovine serum Excell Cat#FSP500; Lot#12C019 Penicillin-Streptomycin Solution, 100X Beyotime Cat#C0222; Lot#A326250724 Insulin procell Cat#PB180432; Lot#WH1724D184 Trypsin-EDTA Solution with phenol red Beyotime Cat#C0204; Lot#Z985241103 Hieff Trans® Liposomal 2000 Transfection Reagent Yeasen Cat#40802ES03; Lot#H3217980 Puromycin Dihydrochloride Beyotime Cat#ST551-50mg; Lot#030424240716 Blasticidin S, Sterile MCE Cat#HY-K1054; Lot#676494 PEI transfection reagent Yeasen Cat#40815ES03; Lot#H5528320 opti-mem Gibco Cat#31985-070; Lot#3141616 Annealing Buffer for DNA Oligos (5X) Beyotime Cat#D0251; Lot#090721220401 AgeI-HF NEB Cat#R3552S; Lot#10126565 EcoRI-HF NEB Cat#R3101S; Lot#10146052 BsmBI-v2 NEB Cat#R0739S; Lot#10170454 T4 DNA Ligase NEB Cat#M0202T; Lot#10126210 Ampicillin Beyotime Cat#ST007; Lot#060221220104 UK-5099 MCE Cat#HY-15475; Lot#07552 (Continued on next page) Cell Reports 45, 117297, May 26, 2026 15 ..
Article Title: AspSnFR: A genetically encoded biosensor for real-time monitoring of aspartate in live cells.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Donkey anti-Goat IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 1mg Thermo Fisher Scientific Inc. Cat#A32849; RRID: AB_2762840 Goat pAb anti HA-tag antibody Novus Bio Cat#NB600-362; RRID: AB_10124937 Bacterial and virus strains BL21(DE3)-pLysS Millipore Cat#69451-3 E. Cloni 10G Lucigen Cat#60117-1 XL1-Blue Agilent Cat#200249 Chemicals, peptides, and recombinant proteins 21 L-amino acids Sigma-Aldrich Cat#LAA21 Ampicillin Sigma-Aldrich Cat#A9393 Antimycin A1 Sigma-Aldrich Cat#A8674 AOA 2-(Aminooxy)Acetic Acid Sigma-Aldrich Cat#C13408 ASCT2 inhibitor WuXi, Wuhan, China N/A BMS-303141 Medchemexpress Cat#HY-16107 Bumetanide Sigma-Aldrich Cat#B3023 Cariporide Sigma-Aldrich Cat#SML1360 CB-839 (Telaglenastat) Medchemexpress Cat#HY-12248 CCCP Sigma-Aldrich Cat#C2759 Cefatriaxone Medchemexpress Cat#HYB-0712B CellLytic M Sigma-Aldrich Cat#C2978 Chloroquine Enamine Cat#EN300-120683 Chlorothiazide ComGenex Ltd, CM-Technology, Budapest, Hungary N/A Cidofovir Waterstone Cat#40997 Compound 968 (GlutaminaseC-IN-1) Sigma-Aldrich Cat#SML1327 Cytochalasin B Cayman Europe Cat#11328 Daporinad (FK866) MedChemExpress Cat#HY-50876 D-aspartate Carl Roth Cat#7873.2 Doxycycline hyclate Sigma-Aldrich Cat#D9891 Furosemide (Desdemin) Enamine Cat#EN300-126738 Gibson assembly mix Home made N/A HCS Cellmask DeepRed Thermo Fisher Scientific Inc. Cat#32721 Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in Water Thermo Fisher Scientific Inc. Cat#H3510 Hydroxychloroquine ABCR Cat#AB564120 Ivosidenib (Tibsovo) MedKoo Biosciences Cat#206148 L-Aspartic Acid (Asp) MerckSigmaAldrich Cat#A9256 Lc-BPE Aurigene, Karnataka 560100, India N/A L-Glutamic Acid (Glu) Sigma-Aldrich Cat#G1251 L-Glutamine (Gln) Sigma-Aldrich Cat#G3126 Losartan ABCR Cat#AB489654 Metformin Sigma-Aldrich Cat#D5035 MitoQ Sigma-Aldrich Cat#ATE959438891 Mitotracker Orange Thermo Fisher Scientific Inc. Cat#M7510 NMN(+) Fluka Cat#72342 (Continued on next page) e1 Cell Chemical Biology 31, 1529–1541.e1–e12, August 15, 2024 .. REAGENT or RESOURCE SOURCE IDENTIFIER Oligomycin A Chemscene Cat#CS-0007502 Omigapil Diverchim SA Cat#DIV00075 Ouabain (Acocantherin) Tocris Cat#1076 Rapamycin (Sirolimus) MedChemExpress Cat#HY-10219 Rheb inhibitor NR-1 WuXi, WuHan China N/A Rotenone TRC Canada Cat#R700580 TFB-TBOA Tocris Cat#2532 Thapsigargin Sigma-Aldrich Cat#T9033 UCPH-101 Sigma-Aldrich Cat#BL3H160B7EB4 Critical commercial assays BsaI-HF v2 NEB Cat#R3733 BsmBI-v2 NEB CatR0739 Pierce BCA Protein Assay Kit Thermo Scientific Inc. Cat#23225 GenElute Mammalian Genomic DNA Miniprep Kit Sigma-Aldrich Cat#G1N350 HisPur Ni-NTA Superflow Agarose Thermo Scientific Inc. Cat#25217 KOD Hot Start Master Mix Sigma-Aldrich Cat#71842 Deposited data SF-iGluSnFR-S72A This paper PDB: 8OVN https://www.rcsb.org/structure/8OVN SF-iGluSnFR-S72A in complex with L-aspartate This paper PDB: 8OVO https://www.rcsb.org/structure/8OVO AspSnFR in complex with L-aspartate This paper PDB: 8OVP https://www.rcsb.org/structure/8OVP Mass spectrometry HCT116SLC25A12-KOOE-AspSnFR This paper MetaboLights: MTBLS10035 https://www.ebi.ac.uk/metabolights/ MTBLS10035 Illumina sequencing data This paper ENA: PRJEB75325 https://www.ebi.ac.uk/ ena/browser/view/PRJEB75325 Transcriptomics (SLC expression) RESOLUTE consortium https://re-solute.eu/knowledgebase/gene ENA: SAMN11893676, SAMN11893683, SAMN11893690 Proteomics (SLC expression) RESOLUTE consortium https://re-solute.eu/knowledgebase/gene ProteomeXchange: PXD051747 Metabolomics (GFP-OE) RESOLUTE consortium https://re-solute.eu/resources/ dashboards/metabolomics/: MetaboLights: MTBLS10077 Experimental models: Cell lines HCT116-SLC25A12-KOOE-AspSnFR This paper N/A HEK293-JI-AspSnFR This paper N/A HEK293-JI-SLC1A3 RESOLUTE consortium RESOLUTE Identifier: CE029W-F https://re-solute.eu/files/ cellline_reports/CE029W-F.pdf Jump In T-REx HEK 293 Kit Thermo Fisher Scientific Inc. Cat#A15008 Oligonculeotides Listed in Table S5 N/A N/A Recombinant DNA pcDNA3.1/cyto-AspSnFr Neomycin This paper Addgene #201394 pcDNA3.1/cyto-AspSnFR Zeocin This paper N/A pET51b(+)/AspSnFR This paper N/A pET51b(+)/AspSnFR lacking mScarlet-I This paper N/A pJTI/cyto-AspSnFR This paper N/A pJTI/R4 Int, Thermo Fisher Scientific Inc. Cat#A15008 pSF-EF1a/EBFP-gpASNase1 This paper N/A (Continued on next page) Cell Chemical Biology 31, 1529–1541.e1–e12, August 15, 2024 e2 .. REAGENT or RESOURCE SOURCE IDENTIFIER pSF-EF1a/gpASNase1 This paper N/A DMS Library, Site-Saturation Variant Library Twist Bioscience N/A Software and algorithms BBtools https://jgi.doe.gov/data-and-tools/ software-tools/bbtools/ https://sourceforge.net/projects/bbmap/ CellProfiler Image analysis Software (OpenSource, Python based) Broad Institute of MIT and Harvard https://cellprofiler.org ChemDraw Professional 22.0 PerkinElmer https://perkinelmer.com FIJI/ImageJ Image processing package (OpenSource, Java based) National Institutes of Health https://imagej.net/ GraphPad Prism 9 GraphPad https://graphpad.com Je demultiplex gbcs.embl.de/Je https://github.com/gbcs-embl/Je JIPipe 1.81 Hans Knöll Institute (HKI), Jena, Germany https://jipipe.hki-jena.de/ Mutagenesis-visualization https://mutagenesis-visualization. readthedocs.io/en/latest/ https://github.com/fhidalgor/ mutagenesis_visualization Other m-Slide 18 Well Ibiditreat Ibidi Cat#81816 m-Slide VI 0.4 Ibidi Cat#80604 384-well plates Corning Cat#3573 96-well plates PerkinElmer Cat#6055260 96-well plates, cell-grade, black, transparent F-bottom BRANDplus Cat#735-2104 Amicon Ultra 4 mL Centrifugal Filters Merck Cat#UFC803024 (30 kDa) Amicon Ultra 4 mL Centrifugal Filters Merck Cat#UFC805024 (50 kDa) Aquamax DW4 microplate washer Molecular Devices LLC.
Article Title: Scalable TCR synthesis and screening enable antigen reactivity mapping in vitiligo.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies APC anti-human CD69 (clone FN50) Biolegend Cat#310910; RRID: AB_314845 PE anti-human CD19 (clone HIB19) Biolegend Cat#302208; RRID: AB_314238 APC anti-human TCR α/β (clone IP26) Biolegend Cat#306718; RRID: AB_10612569 PE anti-human TCR α/β (clone IP26) Biolegend Cat#306708; RRID: AB_314646 Bacterial and virus strains Stbl3 chemical competent cells ThermoFisher Cat#C737303 ElectroMAX® DH10β electrocompetent E. coli ThermoFisher Cat#18290015 Biological samples Suction blister biopsies Vitiligo Clinic and Research Center, University of Massachusetts Chan Medical School N/A Chemicals, peptides, and recombinant proteins Synthesized peptides Genscript Table S2 Phorbol 12-myristate 13-acetate (PMA) ThermoFisher Cat#J63916.MA DEAE-dextran hydrochloride Sigma Cat#D9885-10G Ionomycin, 96% ThermoFisher Cat#J62448.MCR Critical commercial assays NEBNext Ultra II Q5 Master Mix NEB Cat#M0544L KAPA HiFi HotStart Readymix Roche Cat# KK2601 NEBridge Ligase Master Mix NEB Cat#M1100 NEBridge Golden Gate Assembly Kit (BsmBI-v2) NEB CatE1602 NEBridge Golden Gate Assembly Kit (BsaI-HF-v2) NEB Cat#E1601 BsaI-HF-v2 NEB Cat#R3733 BsmBI-v2 NEB Cat#R0739 BbsI-HF NEB Cat#R3539 SapI NEB Cat#R0569 FastDigest XbaI ThermoFisher Cat#FD0685 FastDigest Buffer ThermoFisher Cat#B64 Nuclease-free water (not DEPC-treated) ThermoFisher Cat#AM9937 Shrimp alkaline phosphatase (rSAP) NEB Cat#M0371 ElectroMAX DH10B E. coli ThermoFisher Cat#18290015 MCE membrane filter, 0.025 μm pore size Millipore Sigma Cat#VSWP02500 Chloramphenicol Millipore Sigma Cat#C0378 Electroporation cuvette (1 mm gap) VWR Cat#89047-206 SPRIselect beads Beckman Coulter Cat#B23317 GeneJET Plasmid Miniprep Kit ThermoFisher Cat#K0502 GeneJET Gel Extraction Kit ThermoFisher Cat#K0692 TransIT®-Lenti Transfection Reagent Millipore Sigma Cat#MIR6603 Quikchange Lightning Site-Directed Mutagenesis Kit Agilent Cat#210518 Genscript Genbuilder Gibson Mix Genscript Cat#L00701-50 Ligation sequencing DNA V14 Oxford Nanopore Technologies Cat#SQK-LSK114 Chromium GEM-X Single Cell 5 ′ Kit v3 10X Genomics Cat#PN-1000695 Chromium Next GEM Single Cell 5 ′ Kit v2 10X Genomics Cat#PN-1000263 (Continued on next page) e1 Immunity 59, 1–17.e1–e9, February 10, 2026 .. HEK293T cells (ATCC CRL-11268) were cultured in DMEM (ATCC) supplemented with 10% fetal bovine serum (FBS; Atlanta biologics) and penicillin-streptomycin (Gibco).
Article Title: An AI-driven workflow for the accelerated optimization of cell-free protein synthesis
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-6xHistidin-tag Sigma Cat#SAB2702220; RRID: AB_3095553 anti-GFP-tag Cell signaling Cat#2555; RRID: AB_10692764 anti-HiBiT-tag Promega Cat#N720A Bacterial and virus strains Escherichia coli DH5α Lab stock Escherichia coli BL21 (DE3) Lab stock Escherichia coli W3110 Lab stock Biological samples E. coli cell-free lysates Synthelis Biotech HeLa Lysate ThermoScientific Cat#88882 Chemicals, peptides, and recombinant proteins HEPES Sigma-Aldrich Cat#H3375 Nucleotide mix - Adenosine 5-triphosphate dipotassium salt hydrate Sigma-Aldrich Cat#A8937 Nucleotide mix - Guanosine 5ʹ-Triphosphate, Disodium Salt Sigma-Aldrich Cat#371701 Nucleotide mix - Sel disodique cytidine-5 ′ -triphosphate, 98+ % Thermo Scientific Chemicals Cat#J62238.ME Nucleotide mix - Uridine 5 ′ -triphosphate, sel trisodique, hydraté , 90% Thermo Scientific Chemicals Cat#226310010 3- PGA (Acide D-(− )-3-phosphoglycé rique disodium salt) Sigma-Aldrich Cat#P8877 Spermidine Sigma-Aldrich Cat#85558 Creatine phosphate crystalline, sup à 97% Sigma-Aldrich Cat#10621714001 PEG 8000 Sigma-Aldrich Cat#89510 Mg-glutamate Sigma-Aldrich Cat#49605 K-glutamate Alfa Aesar Cat#A17232 Amino acids - L-Alanine Sigma Cat#A7627 Amino acids - L-Arginine Sigma Cat#A5006 Amino acids - Glycine Sigma Cat#G7126 Amino acids - L-Histidine Merck Cat#1.04351.0100 Amino acids - L-Lysine Sigma Cat#L5626 Amino acids - L-Proline Sigma Cat#P0380 Amino acids - L-Serine Sigma Cat#S4500 Amino acids - L-Threonine Sigma Cat#T8625 Amino acids - L-Valine Sigma Cat#V0500 Amino acids - L-Aspartic Acid Sigma Cat#A9256 Amino acids - L-Asparagine anhydrous Sigma Cat#A0884 Amino acids - L-Cystine Sigma Cat#C8755 Amino acids - L-Glutamine Sigma Cat#G3126 Amino acids - L-Leucine Sigma Cat#L8000 Amino acids - L-Methionine Sigma Cat#64319 Amino acids - L-Tryptophan Sigma Cat#T8941 (Continued on next page) iScience 28, 113599, October 17, 2025 e1 .. REAGENT or RESOURCE SOURCE IDENTIFIER Amino acids - L-Tyrosine Sigma Cat#T3754 Amino acids - L-Phenylalanine Sigma Cat#78019 Amino acids - lsoleucine Sigma Cat#I7403 BsmBI-v2 NEB CatR0739S BbsI NEB Cat#R0539S BsaI NEB Cat#R3733S DpnI NEB Cat#R0176L BamHI Promega Cat#R6021 EcoRV Promega Cat#R6351 T4 DNA ligase Promega Cat#M1801 T4 DNA ligase NEB Cat#M0202 Ampicillin Sigma–Aldrich Cat#A1593 (IPTG) isopropyl -d-1-thiogalactopyranoside Sigma–Aldrich Cat#I5502 Critical commercial assays Q5 High-Fidelity 2X Master Mix NEB Cat#M0492L PCR purification kit NEB Cat#T1130 NucleoBond Xtra Maxi kit Macherey-Nagel Cat#740414.1 Nano-Glo® HiBiT Lytic Detection System Promega Cat#N3030 Nano-Glo® HiBiT Lytic Detection System Promega Cat#N3040 Deposited data icfree-ml source code https://github.com/brsynth/icfree-ml https://doi.org/10.5281/zenodo.14900104 icfree-ml conda packages https://anaconda.org/bioconda/icfree-ml https://doi.org/10.5281/zenodo.14893904 icfree-ml Galaxy wrappers https://toolshed.g2.bx.psu.edu https://doi.org/10.5281/zenodo.14893963 AI-CellFree - Init Galaxy workflow https://galaxy-synbiocad.org/ u/joan/w/icfree-init https://doi.org/10.5281/zenodo.14901023 AI-CellFree - Core Galaxy workflow https://galaxy-synbiocad.org/ u/joan/w/icfree-core https://doi.org/10.5281/zenodo.14899706 Experimental models: Organisms/strains Escherichia coli DH5α Escherichia coli BL21 (DE3) Escherichia coli W3110 Oligonucleotides See Table S3 Recombinant DNA Colicin M Synthesized by GeneCust Colicin E1 Synthesized by GeneCust GFP1-10 Synthesized by GeneCust GFP11 Synthesized by GeneCust His-GFP1-10 This study His-GFP11-Colicin M This study His-GFP11-Colicin E1 This study His-HiBiT-Colicin M This study His-HiBiT-Colicin E1 This study His-HiBiT-sfGFP This study Software and algorithms Echo Cherry Pick software Beckman Coulter Protocol Designer Opentrons Image Lab TM Software Bio-Rad Laboratories ChatGPT-4 Openai e2 iScience 28, 113599, October 17, 2025 ..
Article Title: Protocol to implement saturation mutagenesis-reinforced functional assays to resolve small-sized variants in disease-related genes.
Article Snippet: .. Continued REAGENT or RESOURCE SOURCE IDENTIFIER Viobility 405/452 fixable dye Miltenyi Biotec Cat#130-130-420 MACS BSA stock solution Miltenyi Biotec Cat#130-091-376 autoMACS rinsing solution Miltenyi Biotec Cat#130-091-222 Human BD Fc Block BD Pharmingen Cat#564220 Q5 high-fidelity DNA polymerase NEB Cat#M0491SVIAL Q5 reaction buffer NEB Cat#B9027SVIAL Q5 High GC Enhancer NEB Cat#B9028AVIAL dNTPs NEB Cat#N0447 BsmBI-v2 NEB CatR0739S BsaI-HFv2 NEB Cat#R3733S XbaI NEB Cat#R0145S BamHI-HF NEB Cat#R3136S Type2M enzyme MspJI NEB Cat#R0661S Type2M enzyme DpnI NEB Cat#R0176S Polybrene Sigma-Aldrich Cat#107689 Critical commercial assays Synthetic single guide RNA kit Synthego N/A NucleoSpin gel and PCR clean-up kit Takara Cat#740609 PureLink midiprep kit Invitrogen Cat#K210014 PureLink genomic DNA mini kit Invitrogen Cat#K182002 SE Cell Line 4D-Nucleofector X Kit S Lonza Cat#V4XC-1032 Experimental models: Cell lines HAP1 Horizon Discovery C631 HEK293T ATCC CRL-3216 MB135 Dr. Stephen J. Tapscott N/A Recombinant DNA Lenti-UbC-FKRP-EF1a-BSD Ma et al. 1 Addgene Plasmid #205150 psPAX2 Dr. Didier Trono Addgene Plasmid #12260 pMD2.G Dr. Didier Trono Addgene Plasmid #12259 Software and algorithms R R Core Team https://www.r-project.org/contributors.html Scripts for oligo design, pre-SMuRF experiments Ma et al. 1 https://doi.org/10.5281/zenodo.13328292 Raw NGS data analytical pipeline Ma et al. 1 https://doi.org/10.5281/zenodo.13328302 Scripts for generating SMuRF scores and downstream analyses Ma et al. 1 https://doi.org/10.5281/zenodo.13328286 Other 0.45 μm PES filter Thermo Scientific Cat#165-0045 40 μm cell strainer Falcon Cat#352340 3D Rocker Benchmark Scientific Cat#B3D1008 Nikon microscope object marker Fisher Scientific Cat#NIKON-MBW10020 10 3 10 mm PYREX cloning cylinders Avantor/VWR Cat#22877-256 TC20 automated cell counter Bio-Rad Cat#1450102 Qubit fluorometer Thermo Scientific Cat#Q33226 STAR Protocols 6, 103909, September 19, 2025 3 Note: Synthego Synthetic Single Guide RNA (sgRNA) Kits can be utilized for sgRNA design and synthesis. ..
Transfection:Article Title: SEPHS2 loss reprograms cancer metabolism from oxidative phosphorylation to gluconeogenesis via PCK1 stabilization.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies SEPHS2 proteintech Cat#14109-1-AP; Lot#00124159; RRID: AB_2878014 LA/LP Polyclonal antibody(SEPSECS) proteintech Cat#11551-1-AP; Lot#00121618; RRID:AB_2186853 PCK1 (D12F5) Rabbit mAb Cell Signaling Technology Cat#12940S; Lot#3; RRID:AB_2687968 Anti FLAG Nanobody Agarose Beads ALPALIFEBIO Cat#KTSM1308; Lot#240810; RRID:AB_3740166 Anti-HA tag Rabbit mAb PTM bio Cat#PTM-5389; Lot#RL110916; RRID:AB_3740293 Anti-DYKDDDDK Tag Mouse mAb PTM bio Cat#PTM-6075; Lot#MN120818; RRID:AB_3740294 Pan-Acetyl-Lysine Mab Abmart Cat#M30069S; Lot#334095; RRID: AB_2936273 β-actin Beta Actin Monoclonal antibody proteintech Cat#66009-1-Ig; Lot#10052137; RRID: AB_2687938 HRP-conjugated GAPDH Monoclonal antibody proteintech Cat#HRP-60004; Lot#21020050; RRID: AB_2107436 HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) proteintech Cat#SA00001-2; Lot#20001102; RRID: AB_2722564 HRP-conjugated Affinipure Goat Anti-Mouse IgG(H + L) proteintech Cat#SA00001-1; Lot#20001336; RRID: AB_2722565 Ki-67 Invitrogen Cat#MA5-14520; Lot#ZC4251801; RRID: AB_10979488 BrdU HUA BIO Cat#HA721283; Lot#H651616016; RRID: AB_3072400 Chemicals, peptides, and recombinant proteins DMEM VivaCell Cat#C3113-0500; Lot#2523153 Minimum Essential Medium Eagle,(MEM-E) VivaCell Cat#C3050-0500; Lot#2421230 glucose-free DMEM Wisent Cat#319-061-CL; Lot#319061013 13C6-glucose Cambridge Isotope Laboratories Cat#CLM-1396-1; Lot#PR-34213 Fetal bovine serum Excell Cat#FSP500; Lot#12C019 Penicillin-Streptomycin Solution, 100X Beyotime Cat#C0222; Lot#A326250724 Insulin procell Cat#PB180432; Lot#WH1724D184 Trypsin-EDTA Solution with phenol red Beyotime Cat#C0204; Lot#Z985241103 Hieff Trans® Liposomal 2000 Transfection Reagent Yeasen Cat#40802ES03; Lot#H3217980 Puromycin Dihydrochloride Beyotime Cat#ST551-50mg; Lot#030424240716 Blasticidin S, Sterile MCE Cat#HY-K1054; Lot#676494 PEI transfection reagent Yeasen Cat#40815ES03; Lot#H5528320 opti-mem Gibco Cat#31985-070; Lot#3141616 Annealing Buffer for DNA Oligos (5X) Beyotime Cat#D0251; Lot#090721220401 AgeI-HF NEB Cat#R3552S; Lot#10126565 EcoRI-HF NEB Cat#R3101S; Lot#10146052 BsmBI-v2 NEB Cat#R0739S; Lot#10170454 T4 DNA Ligase NEB Cat#M0202T; Lot#10126210 Ampicillin Beyotime Cat#ST007; Lot#060221220104 UK-5099 MCE Cat#HY-15475; Lot#07552 (Continued on next page) Cell Reports 45, 117297, May 26, 2026 15 ..
Article Title: Scalable TCR synthesis and screening enable antigen reactivity mapping in vitiligo.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies APC anti-human CD69 (clone FN50) Biolegend Cat#310910; RRID: AB_314845 PE anti-human CD19 (clone HIB19) Biolegend Cat#302208; RRID: AB_314238 APC anti-human TCR α/β (clone IP26) Biolegend Cat#306718; RRID: AB_10612569 PE anti-human TCR α/β (clone IP26) Biolegend Cat#306708; RRID: AB_314646 Bacterial and virus strains Stbl3 chemical competent cells ThermoFisher Cat#C737303 ElectroMAX® DH10β electrocompetent E. coli ThermoFisher Cat#18290015 Biological samples Suction blister biopsies Vitiligo Clinic and Research Center, University of Massachusetts Chan Medical School N/A Chemicals, peptides, and recombinant proteins Synthesized peptides Genscript Table S2 Phorbol 12-myristate 13-acetate (PMA) ThermoFisher Cat#J63916.MA DEAE-dextran hydrochloride Sigma Cat#D9885-10G Ionomycin, 96% ThermoFisher Cat#J62448.MCR Critical commercial assays NEBNext Ultra II Q5 Master Mix NEB Cat#M0544L KAPA HiFi HotStart Readymix Roche Cat# KK2601 NEBridge Ligase Master Mix NEB Cat#M1100 NEBridge Golden Gate Assembly Kit (BsmBI-v2) NEB CatE1602 NEBridge Golden Gate Assembly Kit (BsaI-HF-v2) NEB Cat#E1601 BsaI-HF-v2 NEB Cat#R3733 BsmBI-v2 NEB Cat#R0739 BbsI-HF NEB Cat#R3539 SapI NEB Cat#R0569 FastDigest XbaI ThermoFisher Cat#FD0685 FastDigest Buffer ThermoFisher Cat#B64 Nuclease-free water (not DEPC-treated) ThermoFisher Cat#AM9937 Shrimp alkaline phosphatase (rSAP) NEB Cat#M0371 ElectroMAX DH10B E. coli ThermoFisher Cat#18290015 MCE membrane filter, 0.025 μm pore size Millipore Sigma Cat#VSWP02500 Chloramphenicol Millipore Sigma Cat#C0378 Electroporation cuvette (1 mm gap) VWR Cat#89047-206 SPRIselect beads Beckman Coulter Cat#B23317 GeneJET Plasmid Miniprep Kit ThermoFisher Cat#K0502 GeneJET Gel Extraction Kit ThermoFisher Cat#K0692 TransIT®-Lenti Transfection Reagent Millipore Sigma Cat#MIR6603 Quikchange Lightning Site-Directed Mutagenesis Kit Agilent Cat#210518 Genscript Genbuilder Gibson Mix Genscript Cat#L00701-50 Ligation sequencing DNA V14 Oxford Nanopore Technologies Cat#SQK-LSK114 Chromium GEM-X Single Cell 5 ′ Kit v3 10X Genomics Cat#PN-1000695 Chromium Next GEM Single Cell 5 ′ Kit v2 10X Genomics Cat#PN-1000263 (Continued on next page) e1 Immunity 59, 1–17.e1–e9, February 10, 2026 .. HEK293T cells (ATCC CRL-11268) were cultured in DMEM (ATCC) supplemented with 10% fetal bovine serum (FBS; Atlanta biologics) and penicillin-streptomycin (Gibco).
Sterility:Article Title: SEPHS2 loss reprograms cancer metabolism from oxidative phosphorylation to gluconeogenesis via PCK1 stabilization.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies SEPHS2 proteintech Cat#14109-1-AP; Lot#00124159; RRID: AB_2878014 LA/LP Polyclonal antibody(SEPSECS) proteintech Cat#11551-1-AP; Lot#00121618; RRID:AB_2186853 PCK1 (D12F5) Rabbit mAb Cell Signaling Technology Cat#12940S; Lot#3; RRID:AB_2687968 Anti FLAG Nanobody Agarose Beads ALPALIFEBIO Cat#KTSM1308; Lot#240810; RRID:AB_3740166 Anti-HA tag Rabbit mAb PTM bio Cat#PTM-5389; Lot#RL110916; RRID:AB_3740293 Anti-DYKDDDDK Tag Mouse mAb PTM bio Cat#PTM-6075; Lot#MN120818; RRID:AB_3740294 Pan-Acetyl-Lysine Mab Abmart Cat#M30069S; Lot#334095; RRID: AB_2936273 β-actin Beta Actin Monoclonal antibody proteintech Cat#66009-1-Ig; Lot#10052137; RRID: AB_2687938 HRP-conjugated GAPDH Monoclonal antibody proteintech Cat#HRP-60004; Lot#21020050; RRID: AB_2107436 HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) proteintech Cat#SA00001-2; Lot#20001102; RRID: AB_2722564 HRP-conjugated Affinipure Goat Anti-Mouse IgG(H + L) proteintech Cat#SA00001-1; Lot#20001336; RRID: AB_2722565 Ki-67 Invitrogen Cat#MA5-14520; Lot#ZC4251801; RRID: AB_10979488 BrdU HUA BIO Cat#HA721283; Lot#H651616016; RRID: AB_3072400 Chemicals, peptides, and recombinant proteins DMEM VivaCell Cat#C3113-0500; Lot#2523153 Minimum Essential Medium Eagle,(MEM-E) VivaCell Cat#C3050-0500; Lot#2421230 glucose-free DMEM Wisent Cat#319-061-CL; Lot#319061013 13C6-glucose Cambridge Isotope Laboratories Cat#CLM-1396-1; Lot#PR-34213 Fetal bovine serum Excell Cat#FSP500; Lot#12C019 Penicillin-Streptomycin Solution, 100X Beyotime Cat#C0222; Lot#A326250724 Insulin procell Cat#PB180432; Lot#WH1724D184 Trypsin-EDTA Solution with phenol red Beyotime Cat#C0204; Lot#Z985241103 Hieff Trans® Liposomal 2000 Transfection Reagent Yeasen Cat#40802ES03; Lot#H3217980 Puromycin Dihydrochloride Beyotime Cat#ST551-50mg; Lot#030424240716 Blasticidin S, Sterile MCE Cat#HY-K1054; Lot#676494 PEI transfection reagent Yeasen Cat#40815ES03; Lot#H5528320 opti-mem Gibco Cat#31985-070; Lot#3141616 Annealing Buffer for DNA Oligos (5X) Beyotime Cat#D0251; Lot#090721220401 AgeI-HF NEB Cat#R3552S; Lot#10126565 EcoRI-HF NEB Cat#R3101S; Lot#10146052 BsmBI-v2 NEB Cat#R0739S; Lot#10170454 T4 DNA Ligase NEB Cat#M0202T; Lot#10126210 Ampicillin Beyotime Cat#ST007; Lot#060221220104 UK-5099 MCE Cat#HY-15475; Lot#07552 (Continued on next page) Cell Reports 45, 117297, May 26, 2026 15 ..
Virus:
Article Title: Scalable TCR synthesis and screening enable antigen reactivity mapping in vitiligo.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies APC anti-human CD69 (clone FN50) Biolegend Cat#310910; RRID: AB_314845 PE anti-human CD19 (clone HIB19) Biolegend Cat#302208; RRID: AB_314238 APC anti-human TCR α/β (clone IP26) Biolegend Cat#306718; RRID: AB_10612569 PE anti-human TCR α/β (clone IP26) Biolegend Cat#306708; RRID: AB_314646 Bacterial and virus strains Stbl3 chemical competent cells ThermoFisher Cat#C737303 ElectroMAX® DH10β electrocompetent E. coli ThermoFisher Cat#18290015 Biological samples Suction blister biopsies Vitiligo Clinic and Research Center, University of Massachusetts Chan Medical School N/A Chemicals, peptides, and recombinant proteins Synthesized peptides Genscript Table S2 Phorbol 12-myristate 13-acetate (PMA) ThermoFisher Cat#J63916.MA DEAE-dextran hydrochloride Sigma Cat#D9885-10G Ionomycin, 96% ThermoFisher Cat#J62448.MCR Critical commercial assays NEBNext Ultra II Q5 Master Mix NEB Cat#M0544L KAPA HiFi HotStart Readymix Roche Cat# KK2601 NEBridge Ligase Master Mix NEB Cat#M1100 NEBridge Golden Gate Assembly Kit (BsmBI-v2) NEB CatE1602 NEBridge Golden Gate Assembly Kit (BsaI-HF-v2) NEB Cat#E1601 BsaI-HF-v2 NEB Cat#R3733 BsmBI-v2 NEB Cat#R0739 BbsI-HF NEB Cat#R3539 SapI NEB Cat#R0569 FastDigest XbaI ThermoFisher Cat#FD0685 FastDigest Buffer ThermoFisher Cat#B64 Nuclease-free water (not DEPC-treated) ThermoFisher Cat#AM9937 Shrimp alkaline phosphatase (rSAP) NEB Cat#M0371 ElectroMAX DH10B E. coli ThermoFisher Cat#18290015 MCE membrane filter, 0.025 μm pore size Millipore Sigma Cat#VSWP02500 Chloramphenicol Millipore Sigma Cat#C0378 Electroporation cuvette (1 mm gap) VWR Cat#89047-206 SPRIselect beads Beckman Coulter Cat#B23317 GeneJET Plasmid Miniprep Kit ThermoFisher Cat#K0502 GeneJET Gel Extraction Kit ThermoFisher Cat#K0692 TransIT®-Lenti Transfection Reagent Millipore Sigma Cat#MIR6603 Quikchange Lightning Site-Directed Mutagenesis Kit Agilent Cat#210518 Genscript Genbuilder Gibson Mix Genscript Cat#L00701-50 Ligation sequencing DNA V14 Oxford Nanopore Technologies Cat#SQK-LSK114 Chromium GEM-X Single Cell 5 ′ Kit v3 10X Genomics Cat#PN-1000695 Chromium Next GEM Single Cell 5 ′ Kit v2 10X Genomics Cat#PN-1000263 (Continued on next page) e1 Immunity 59, 1–17.e1–e9, February 10, 2026 .. HEK293T cells (ATCC CRL-11268) were cultured in DMEM (ATCC) supplemented with 10% fetal bovine serum (FBS; Atlanta biologics) and penicillin-streptomycin (Gibco).
Mutagenesis:
Article Title: Scalable TCR synthesis and screening enable antigen reactivity mapping in vitiligo.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies APC anti-human CD69 (clone FN50) Biolegend Cat#310910; RRID: AB_314845 PE anti-human CD19 (clone HIB19) Biolegend Cat#302208; RRID: AB_314238 APC anti-human TCR α/β (clone IP26) Biolegend Cat#306718; RRID: AB_10612569 PE anti-human TCR α/β (clone IP26) Biolegend Cat#306708; RRID: AB_314646 Bacterial and virus strains Stbl3 chemical competent cells ThermoFisher Cat#C737303 ElectroMAX® DH10β electrocompetent E. coli ThermoFisher Cat#18290015 Biological samples Suction blister biopsies Vitiligo Clinic and Research Center, University of Massachusetts Chan Medical School N/A Chemicals, peptides, and recombinant proteins Synthesized peptides Genscript Table S2 Phorbol 12-myristate 13-acetate (PMA) ThermoFisher Cat#J63916.MA DEAE-dextran hydrochloride Sigma Cat#D9885-10G Ionomycin, 96% ThermoFisher Cat#J62448.MCR Critical commercial assays NEBNext Ultra II Q5 Master Mix NEB Cat#M0544L KAPA HiFi HotStart Readymix Roche Cat# KK2601 NEBridge Ligase Master Mix NEB Cat#M1100 NEBridge Golden Gate Assembly Kit (BsmBI-v2) NEB CatE1602 NEBridge Golden Gate Assembly Kit (BsaI-HF-v2) NEB Cat#E1601 BsaI-HF-v2 NEB Cat#R3733 BsmBI-v2 NEB Cat#R0739 BbsI-HF NEB Cat#R3539 SapI NEB Cat#R0569 FastDigest XbaI ThermoFisher Cat#FD0685 FastDigest Buffer ThermoFisher Cat#B64 Nuclease-free water (not DEPC-treated) ThermoFisher Cat#AM9937 Shrimp alkaline phosphatase (rSAP) NEB Cat#M0371 ElectroMAX DH10B E. coli ThermoFisher Cat#18290015 MCE membrane filter, 0.025 μm pore size Millipore Sigma Cat#VSWP02500 Chloramphenicol Millipore Sigma Cat#C0378 Electroporation cuvette (1 mm gap) VWR Cat#89047-206 SPRIselect beads Beckman Coulter Cat#B23317 GeneJET Plasmid Miniprep Kit ThermoFisher Cat#K0502 GeneJET Gel Extraction Kit ThermoFisher Cat#K0692 TransIT®-Lenti Transfection Reagent Millipore Sigma Cat#MIR6603 Quikchange Lightning Site-Directed Mutagenesis Kit Agilent Cat#210518 Genscript Genbuilder Gibson Mix Genscript Cat#L00701-50 Ligation sequencing DNA V14 Oxford Nanopore Technologies Cat#SQK-LSK114 Chromium GEM-X Single Cell 5 ′ Kit v3 10X Genomics Cat#PN-1000695 Chromium Next GEM Single Cell 5 ′ Kit v2 10X Genomics Cat#PN-1000263 (Continued on next page) e1 Immunity 59, 1–17.e1–e9, February 10, 2026 .. HEK293T cells (ATCC CRL-11268) were cultured in DMEM (ATCC) supplemented with 10% fetal bovine serum (FBS; Atlanta biologics) and penicillin-streptomycin (Gibco).
Reverse Transcription:
Polymerase Chain Reaction:
Article Title: An AI-driven workflow for the accelerated optimization of cell-free protein synthesis
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-6xHistidin-tag Sigma Cat#SAB2702220; RRID: AB_3095553 anti-GFP-tag Cell signaling Cat#2555; RRID: AB_10692764 anti-HiBiT-tag Promega Cat#N720A Bacterial and virus strains Escherichia coli DH5α Lab stock Escherichia coli BL21 (DE3) Lab stock Escherichia coli W3110 Lab stock Biological samples E. coli cell-free lysates Synthelis Biotech HeLa Lysate ThermoScientific Cat#88882 Chemicals, peptides, and recombinant proteins HEPES Sigma-Aldrich Cat#H3375 Nucleotide mix - Adenosine 5-triphosphate dipotassium salt hydrate Sigma-Aldrich Cat#A8937 Nucleotide mix - Guanosine 5ʹ-Triphosphate, Disodium Salt Sigma-Aldrich Cat#371701 Nucleotide mix - Sel disodique cytidine-5 ′ -triphosphate, 98+ % Thermo Scientific Chemicals Cat#J62238.ME Nucleotide mix - Uridine 5 ′ -triphosphate, sel trisodique, hydraté , 90% Thermo Scientific Chemicals Cat#226310010 3- PGA (Acide D-(− )-3-phosphoglycé rique disodium salt) Sigma-Aldrich Cat#P8877 Spermidine Sigma-Aldrich Cat#85558 Creatine phosphate crystalline, sup à 97% Sigma-Aldrich Cat#10621714001 PEG 8000 Sigma-Aldrich Cat#89510 Mg-glutamate Sigma-Aldrich Cat#49605 K-glutamate Alfa Aesar Cat#A17232 Amino acids - L-Alanine Sigma Cat#A7627 Amino acids - L-Arginine Sigma Cat#A5006 Amino acids - Glycine Sigma Cat#G7126 Amino acids - L-Histidine Merck Cat#1.04351.0100 Amino acids - L-Lysine Sigma Cat#L5626 Amino acids - L-Proline Sigma Cat#P0380 Amino acids - L-Serine Sigma Cat#S4500 Amino acids - L-Threonine Sigma Cat#T8625 Amino acids - L-Valine Sigma Cat#V0500 Amino acids - L-Aspartic Acid Sigma Cat#A9256 Amino acids - L-Asparagine anhydrous Sigma Cat#A0884 Amino acids - L-Cystine Sigma Cat#C8755 Amino acids - L-Glutamine Sigma Cat#G3126 Amino acids - L-Leucine Sigma Cat#L8000 Amino acids - L-Methionine Sigma Cat#64319 Amino acids - L-Tryptophan Sigma Cat#T8941 (Continued on next page) iScience 28, 113599, October 17, 2025 e1 .. REAGENT or RESOURCE SOURCE IDENTIFIER Amino acids - L-Tyrosine Sigma Cat#T3754 Amino acids - L-Phenylalanine Sigma Cat#78019 Amino acids - lsoleucine Sigma Cat#I7403 BsmBI-v2 NEB CatR0739S BbsI NEB Cat#R0539S BsaI NEB Cat#R3733S DpnI NEB Cat#R0176L BamHI Promega Cat#R6021 EcoRV Promega Cat#R6351 T4 DNA ligase Promega Cat#M1801 T4 DNA ligase NEB Cat#M0202 Ampicillin Sigma–Aldrich Cat#A1593 (IPTG) isopropyl -d-1-thiogalactopyranoside Sigma–Aldrich Cat#I5502 Critical commercial assays Q5 High-Fidelity 2X Master Mix NEB Cat#M0492L PCR purification kit NEB Cat#T1130 NucleoBond Xtra Maxi kit Macherey-Nagel Cat#740414.1 Nano-Glo® HiBiT Lytic Detection System Promega Cat#N3030 Nano-Glo® HiBiT Lytic Detection System Promega Cat#N3040 Deposited data icfree-ml source code https://github.com/brsynth/icfree-ml https://doi.org/10.5281/zenodo.14900104 icfree-ml conda packages https://anaconda.org/bioconda/icfree-ml https://doi.org/10.5281/zenodo.14893904 icfree-ml Galaxy wrappers https://toolshed.g2.bx.psu.edu https://doi.org/10.5281/zenodo.14893963 AI-CellFree - Init Galaxy workflow https://galaxy-synbiocad.org/ u/joan/w/icfree-init https://doi.org/10.5281/zenodo.14901023 AI-CellFree - Core Galaxy workflow https://galaxy-synbiocad.org/ u/joan/w/icfree-core https://doi.org/10.5281/zenodo.14899706 Experimental models: Organisms/strains Escherichia coli DH5α Escherichia coli BL21 (DE3) Escherichia coli W3110 Oligonucleotides See Table S3 Recombinant DNA Colicin M Synthesized by GeneCust Colicin E1 Synthesized by GeneCust GFP1-10 Synthesized by GeneCust GFP11 Synthesized by GeneCust His-GFP1-10 This study His-GFP11-Colicin M This study His-GFP11-Colicin E1 This study His-HiBiT-Colicin M This study His-HiBiT-Colicin E1 This study His-HiBiT-sfGFP This study Software and algorithms Echo Cherry Pick software Beckman Coulter Protocol Designer Opentrons Image Lab TM Software Bio-Rad Laboratories ChatGPT-4 Openai e2 iScience 28, 113599, October 17, 2025 ..
Article Title: Protocol to implement saturation mutagenesis-reinforced functional assays to resolve small-sized variants in disease-related genes.
Article Snippet: .. Continued REAGENT or RESOURCE SOURCE IDENTIFIER Viobility 405/452 fixable dye Miltenyi Biotec Cat#130-130-420 MACS BSA stock solution Miltenyi Biotec Cat#130-091-376 autoMACS rinsing solution Miltenyi Biotec Cat#130-091-222 Human BD Fc Block BD Pharmingen Cat#564220 Q5 high-fidelity DNA polymerase NEB Cat#M0491SVIAL Q5 reaction buffer NEB Cat#B9027SVIAL Q5 High GC Enhancer NEB Cat#B9028AVIAL dNTPs NEB Cat#N0447 BsmBI-v2 NEB CatR0739S BsaI-HFv2 NEB Cat#R3733S XbaI NEB Cat#R0145S BamHI-HF NEB Cat#R3136S Type2M enzyme MspJI NEB Cat#R0661S Type2M enzyme DpnI NEB Cat#R0176S Polybrene Sigma-Aldrich Cat#107689 Critical commercial assays Synthetic single guide RNA kit Synthego N/A NucleoSpin gel and PCR clean-up kit Takara Cat#740609 PureLink midiprep kit Invitrogen Cat#K210014 PureLink genomic DNA mini kit Invitrogen Cat#K182002 SE Cell Line 4D-Nucleofector X Kit S Lonza Cat#V4XC-1032 Experimental models: Cell lines HAP1 Horizon Discovery C631 HEK293T ATCC CRL-3216 MB135 Dr. Stephen J. Tapscott N/A Recombinant DNA Lenti-UbC-FKRP-EF1a-BSD Ma et al. 1 Addgene Plasmid #205150 psPAX2 Dr. Didier Trono Addgene Plasmid #12260 pMD2.G Dr. Didier Trono Addgene Plasmid #12259 Software and algorithms R R Core Team https://www.r-project.org/contributors.html Scripts for oligo design, pre-SMuRF experiments Ma et al. 1 https://doi.org/10.5281/zenodo.13328292 Raw NGS data analytical pipeline Ma et al. 1 https://doi.org/10.5281/zenodo.13328302 Scripts for generating SMuRF scores and downstream analyses Ma et al. 1 https://doi.org/10.5281/zenodo.13328286 Other 0.45 μm PES filter Thermo Scientific Cat#165-0045 40 μm cell strainer Falcon Cat#352340 3D Rocker Benchmark Scientific Cat#B3D1008 Nikon microscope object marker Fisher Scientific Cat#NIKON-MBW10020 10 3 10 mm PYREX cloning cylinders Avantor/VWR Cat#22877-256 TC20 automated cell counter Bio-Rad Cat#1450102 Qubit fluorometer Thermo Scientific Cat#Q33226 STAR Protocols 6, 103909, September 19, 2025 3 Note: Synthego Synthetic Single Guide RNA (sgRNA) Kits can be utilized for sgRNA design and synthesis. ..
Magnetic Cell Separation:
Article Title: Protocol to implement saturation mutagenesis-reinforced functional assays to resolve small-sized variants in disease-related genes.
Article Snippet: .. Continued REAGENT or RESOURCE SOURCE IDENTIFIER Viobility 405/452 fixable dye Miltenyi Biotec Cat#130-130-420 MACS BSA stock solution Miltenyi Biotec Cat#130-091-376 autoMACS rinsing solution Miltenyi Biotec Cat#130-091-222 Human BD Fc Block BD Pharmingen Cat#564220 Q5 high-fidelity DNA polymerase NEB Cat#M0491SVIAL Q5 reaction buffer NEB Cat#B9027SVIAL Q5 High GC Enhancer NEB Cat#B9028AVIAL dNTPs NEB Cat#N0447 BsmBI-v2 NEB CatR0739S BsaI-HFv2 NEB Cat#R3733S XbaI NEB Cat#R0145S BamHI-HF NEB Cat#R3136S Type2M enzyme MspJI NEB Cat#R0661S Type2M enzyme DpnI NEB Cat#R0176S Polybrene Sigma-Aldrich Cat#107689 Critical commercial assays Synthetic single guide RNA kit Synthego N/A NucleoSpin gel and PCR clean-up kit Takara Cat#740609 PureLink midiprep kit Invitrogen Cat#K210014 PureLink genomic DNA mini kit Invitrogen Cat#K182002 SE Cell Line 4D-Nucleofector X Kit S Lonza Cat#V4XC-1032 Experimental models: Cell lines HAP1 Horizon Discovery C631 HEK293T ATCC CRL-3216 MB135 Dr. Stephen J. Tapscott N/A Recombinant DNA Lenti-UbC-FKRP-EF1a-BSD Ma et al. 1 Addgene Plasmid #205150 psPAX2 Dr. Didier Trono Addgene Plasmid #12260 pMD2.G Dr. Didier Trono Addgene Plasmid #12259 Software and algorithms R R Core Team https://www.r-project.org/contributors.html Scripts for oligo design, pre-SMuRF experiments Ma et al. 1 https://doi.org/10.5281/zenodo.13328292 Raw NGS data analytical pipeline Ma et al. 1 https://doi.org/10.5281/zenodo.13328302 Scripts for generating SMuRF scores and downstream analyses Ma et al. 1 https://doi.org/10.5281/zenodo.13328286 Other 0.45 μm PES filter Thermo Scientific Cat#165-0045 40 μm cell strainer Falcon Cat#352340 3D Rocker Benchmark Scientific Cat#B3D1008 Nikon microscope object marker Fisher Scientific Cat#NIKON-MBW10020 10 3 10 mm PYREX cloning cylinders Avantor/VWR Cat#22877-256 TC20 automated cell counter Bio-Rad Cat#1450102 Qubit fluorometer Thermo Scientific Cat#Q33226 STAR Protocols 6, 103909, September 19, 2025 3 Note: Synthego Synthetic Single Guide RNA (sgRNA) Kits can be utilized for sgRNA design and synthesis. ..
Cloning:
Article Title: Protocol to implement saturation mutagenesis-reinforced functional assays to resolve small-sized variants in disease-related genes.
Article Snippet: .. Continued REAGENT or RESOURCE SOURCE IDENTIFIER Viobility 405/452 fixable dye Miltenyi Biotec Cat#130-130-420 MACS BSA stock solution Miltenyi Biotec Cat#130-091-376 autoMACS rinsing solution Miltenyi Biotec Cat#130-091-222 Human BD Fc Block BD Pharmingen Cat#564220 Q5 high-fidelity DNA polymerase NEB Cat#M0491SVIAL Q5 reaction buffer NEB Cat#B9027SVIAL Q5 High GC Enhancer NEB Cat#B9028AVIAL dNTPs NEB Cat#N0447 BsmBI-v2 NEB CatR0739S BsaI-HFv2 NEB Cat#R3733S XbaI NEB Cat#R0145S BamHI-HF NEB Cat#R3136S Type2M enzyme MspJI NEB Cat#R0661S Type2M enzyme DpnI NEB Cat#R0176S Polybrene Sigma-Aldrich Cat#107689 Critical commercial assays Synthetic single guide RNA kit Synthego N/A NucleoSpin gel and PCR clean-up kit Takara Cat#740609 PureLink midiprep kit Invitrogen Cat#K210014 PureLink genomic DNA mini kit Invitrogen Cat#K182002 SE Cell Line 4D-Nucleofector X Kit S Lonza Cat#V4XC-1032 Experimental models: Cell lines HAP1 Horizon Discovery C631 HEK293T ATCC CRL-3216 MB135 Dr. Stephen J. Tapscott N/A Recombinant DNA Lenti-UbC-FKRP-EF1a-BSD Ma et al. 1 Addgene Plasmid #205150 psPAX2 Dr. Didier Trono Addgene Plasmid #12260 pMD2.G Dr. Didier Trono Addgene Plasmid #12259 Software and algorithms R R Core Team https://www.r-project.org/contributors.html Scripts for oligo design, pre-SMuRF experiments Ma et al. 1 https://doi.org/10.5281/zenodo.13328292 Raw NGS data analytical pipeline Ma et al. 1 https://doi.org/10.5281/zenodo.13328302 Scripts for generating SMuRF scores and downstream analyses Ma et al. 1 https://doi.org/10.5281/zenodo.13328286 Other 0.45 μm PES filter Thermo Scientific Cat#165-0045 40 μm cell strainer Falcon Cat#352340 3D Rocker Benchmark Scientific Cat#B3D1008 Nikon microscope object marker Fisher Scientific Cat#NIKON-MBW10020 10 3 10 mm PYREX cloning cylinders Avantor/VWR Cat#22877-256 TC20 automated cell counter Bio-Rad Cat#1450102 Qubit fluorometer Thermo Scientific Cat#Q33226 STAR Protocols 6, 103909, September 19, 2025 3 Note: Synthego Synthetic Single Guide RNA (sgRNA) Kits can be utilized for sgRNA design and synthesis. ..
CCK-8 Assay:
Lysis:
Acetylene Reduction Assay:
Mass Spectrometry:
Article Title: AspSnFR: A genetically encoded biosensor for real-time monitoring of aspartate in live cells.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Donkey anti-Goat IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 1mg Thermo Fisher Scientific Inc. Cat#A32849; RRID: AB_2762840 Goat pAb anti HA-tag antibody Novus Bio Cat#NB600-362; RRID: AB_10124937 Bacterial and virus strains BL21(DE3)-pLysS Millipore Cat#69451-3 E. Cloni 10G Lucigen Cat#60117-1 XL1-Blue Agilent Cat#200249 Chemicals, peptides, and recombinant proteins 21 L-amino acids Sigma-Aldrich Cat#LAA21 Ampicillin Sigma-Aldrich Cat#A9393 Antimycin A1 Sigma-Aldrich Cat#A8674 AOA 2-(Aminooxy)Acetic Acid Sigma-Aldrich Cat#C13408 ASCT2 inhibitor WuXi, Wuhan, China N/A BMS-303141 Medchemexpress Cat#HY-16107 Bumetanide Sigma-Aldrich Cat#B3023 Cariporide Sigma-Aldrich Cat#SML1360 CB-839 (Telaglenastat) Medchemexpress Cat#HY-12248 CCCP Sigma-Aldrich Cat#C2759 Cefatriaxone Medchemexpress Cat#HYB-0712B CellLytic M Sigma-Aldrich Cat#C2978 Chloroquine Enamine Cat#EN300-120683 Chlorothiazide ComGenex Ltd, CM-Technology, Budapest, Hungary N/A Cidofovir Waterstone Cat#40997 Compound 968 (GlutaminaseC-IN-1) Sigma-Aldrich Cat#SML1327 Cytochalasin B Cayman Europe Cat#11328 Daporinad (FK866) MedChemExpress Cat#HY-50876 D-aspartate Carl Roth Cat#7873.2 Doxycycline hyclate Sigma-Aldrich Cat#D9891 Furosemide (Desdemin) Enamine Cat#EN300-126738 Gibson assembly mix Home made N/A HCS Cellmask DeepRed Thermo Fisher Scientific Inc. Cat#32721 Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in Water Thermo Fisher Scientific Inc. Cat#H3510 Hydroxychloroquine ABCR Cat#AB564120 Ivosidenib (Tibsovo) MedKoo Biosciences Cat#206148 L-Aspartic Acid (Asp) MerckSigmaAldrich Cat#A9256 Lc-BPE Aurigene, Karnataka 560100, India N/A L-Glutamic Acid (Glu) Sigma-Aldrich Cat#G1251 L-Glutamine (Gln) Sigma-Aldrich Cat#G3126 Losartan ABCR Cat#AB489654 Metformin Sigma-Aldrich Cat#D5035 MitoQ Sigma-Aldrich Cat#ATE959438891 Mitotracker Orange Thermo Fisher Scientific Inc. Cat#M7510 NMN(+) Fluka Cat#72342 (Continued on next page) e1 Cell Chemical Biology 31, 1529–1541.e1–e12, August 15, 2024 .. REAGENT or RESOURCE SOURCE IDENTIFIER Oligomycin A Chemscene Cat#CS-0007502 Omigapil Diverchim SA Cat#DIV00075 Ouabain (Acocantherin) Tocris Cat#1076 Rapamycin (Sirolimus) MedChemExpress Cat#HY-10219 Rheb inhibitor NR-1 WuXi, WuHan China N/A Rotenone TRC Canada Cat#R700580 TFB-TBOA Tocris Cat#2532 Thapsigargin Sigma-Aldrich Cat#T9033 UCPH-101 Sigma-Aldrich Cat#BL3H160B7EB4 Critical commercial assays BsaI-HF v2 NEB Cat#R3733 BsmBI-v2 NEB CatR0739 Pierce BCA Protein Assay Kit Thermo Scientific Inc. Cat#23225 GenElute Mammalian Genomic DNA Miniprep Kit Sigma-Aldrich Cat#G1N350 HisPur Ni-NTA Superflow Agarose Thermo Scientific Inc. Cat#25217 KOD Hot Start Master Mix Sigma-Aldrich Cat#71842 Deposited data SF-iGluSnFR-S72A This paper PDB: 8OVN https://www.rcsb.org/structure/8OVN SF-iGluSnFR-S72A in complex with L-aspartate This paper PDB: 8OVO https://www.rcsb.org/structure/8OVO AspSnFR in complex with L-aspartate This paper PDB: 8OVP https://www.rcsb.org/structure/8OVP Mass spectrometry HCT116SLC25A12-KOOE-AspSnFR This paper MetaboLights: MTBLS10035 https://www.ebi.ac.uk/metabolights/ MTBLS10035 Illumina sequencing data This paper ENA: PRJEB75325 https://www.ebi.ac.uk/ ena/browser/view/PRJEB75325 Transcriptomics (SLC expression) RESOLUTE consortium https://re-solute.eu/knowledgebase/gene ENA: SAMN11893676, SAMN11893683, SAMN11893690 Proteomics (SLC expression) RESOLUTE consortium https://re-solute.eu/knowledgebase/gene ProteomeXchange: PXD051747 Metabolomics (GFP-OE) RESOLUTE consortium https://re-solute.eu/resources/ dashboards/metabolomics/: MetaboLights: MTBLS10077 Experimental models: Cell lines HCT116-SLC25A12-KOOE-AspSnFR This paper N/A HEK293-JI-AspSnFR This paper N/A HEK293-JI-SLC1A3 RESOLUTE consortium RESOLUTE Identifier: CE029W-F https://re-solute.eu/files/ cellline_reports/CE029W-F.pdf Jump In T-REx HEK 293 Kit Thermo Fisher Scientific Inc. Cat#A15008 Oligonculeotides Listed in Table S5 N/A N/A Recombinant DNA pcDNA3.1/cyto-AspSnFr Neomycin This paper Addgene #201394 pcDNA3.1/cyto-AspSnFR Zeocin This paper N/A pET51b(+)/AspSnFR This paper N/A pET51b(+)/AspSnFR lacking mScarlet-I This paper N/A pJTI/cyto-AspSnFR This paper N/A pJTI/R4 Int, Thermo Fisher Scientific Inc. Cat#A15008 pSF-EF1a/EBFP-gpASNase1 This paper N/A (Continued on next page) Cell Chemical Biology 31, 1529–1541.e1–e12, August 15, 2024 e2 .. REAGENT or RESOURCE SOURCE IDENTIFIER pSF-EF1a/gpASNase1 This paper N/A DMS Library, Site-Saturation Variant Library Twist Bioscience N/A Software and algorithms BBtools https://jgi.doe.gov/data-and-tools/ software-tools/bbtools/ https://sourceforge.net/projects/bbmap/ CellProfiler Image analysis Software (OpenSource, Python based) Broad Institute of MIT and Harvard https://cellprofiler.org ChemDraw Professional 22.0 PerkinElmer https://perkinelmer.com FIJI/ImageJ Image processing package (OpenSource, Java based) National Institutes of Health https://imagej.net/ GraphPad Prism 9 GraphPad https://graphpad.com Je demultiplex gbcs.embl.de/Je https://github.com/gbcs-embl/Je JIPipe 1.81 Hans Knöll Institute (HKI), Jena, Germany https://jipipe.hki-jena.de/ Mutagenesis-visualization https://mutagenesis-visualization. readthedocs.io/en/latest/ https://github.com/fhidalgor/ mutagenesis_visualization Other m-Slide 18 Well Ibiditreat Ibidi Cat#81816 m-Slide VI 0.4 Ibidi Cat#80604 384-well plates Corning Cat#3573 96-well plates PerkinElmer Cat#6055260 96-well plates, cell-grade, black, transparent F-bottom BRANDplus Cat#735-2104 Amicon Ultra 4 mL Centrifugal Filters Merck Cat#UFC803024 (30 kDa) Amicon Ultra 4 mL Centrifugal Filters Merck Cat#UFC805024 (50 kDa) Aquamax DW4 microplate washer Molecular Devices LLC.
Liquid Chromatography with Mass Spectroscopy:
Real-time Polymerase Chain Reaction:
Bicinchoninic Acid Protein Assay:Article Title: AspSnFR: A genetically encoded biosensor for real-time monitoring of aspartate in live cells.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Donkey anti-Goat IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 1mg Thermo Fisher Scientific Inc. Cat#A32849; RRID: AB_2762840 Goat pAb anti HA-tag antibody Novus Bio Cat#NB600-362; RRID: AB_10124937 Bacterial and virus strains BL21(DE3)-pLysS Millipore Cat#69451-3 E. Cloni 10G Lucigen Cat#60117-1 XL1-Blue Agilent Cat#200249 Chemicals, peptides, and recombinant proteins 21 L-amino acids Sigma-Aldrich Cat#LAA21 Ampicillin Sigma-Aldrich Cat#A9393 Antimycin A1 Sigma-Aldrich Cat#A8674 AOA 2-(Aminooxy)Acetic Acid Sigma-Aldrich Cat#C13408 ASCT2 inhibitor WuXi, Wuhan, China N/A BMS-303141 Medchemexpress Cat#HY-16107 Bumetanide Sigma-Aldrich Cat#B3023 Cariporide Sigma-Aldrich Cat#SML1360 CB-839 (Telaglenastat) Medchemexpress Cat#HY-12248 CCCP Sigma-Aldrich Cat#C2759 Cefatriaxone Medchemexpress Cat#HYB-0712B CellLytic M Sigma-Aldrich Cat#C2978 Chloroquine Enamine Cat#EN300-120683 Chlorothiazide ComGenex Ltd, CM-Technology, Budapest, Hungary N/A Cidofovir Waterstone Cat#40997 Compound 968 (GlutaminaseC-IN-1) Sigma-Aldrich Cat#SML1327 Cytochalasin B Cayman Europe Cat#11328 Daporinad (FK866) MedChemExpress Cat#HY-50876 D-aspartate Carl Roth Cat#7873.2 Doxycycline hyclate Sigma-Aldrich Cat#D9891 Furosemide (Desdemin) Enamine Cat#EN300-126738 Gibson assembly mix Home made N/A HCS Cellmask DeepRed Thermo Fisher Scientific Inc. Cat#32721 Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in Water Thermo Fisher Scientific Inc. Cat#H3510 Hydroxychloroquine ABCR Cat#AB564120 Ivosidenib (Tibsovo) MedKoo Biosciences Cat#206148 L-Aspartic Acid (Asp) MerckSigmaAldrich Cat#A9256 Lc-BPE Aurigene, Karnataka 560100, India N/A L-Glutamic Acid (Glu) Sigma-Aldrich Cat#G1251 L-Glutamine (Gln) Sigma-Aldrich Cat#G3126 Losartan ABCR Cat#AB489654 Metformin Sigma-Aldrich Cat#D5035 MitoQ Sigma-Aldrich Cat#ATE959438891 Mitotracker Orange Thermo Fisher Scientific Inc. Cat#M7510 NMN(+) Fluka Cat#72342 (Continued on next page) e1 Cell Chemical Biology 31, 1529–1541.e1–e12, August 15, 2024 .. REAGENT or RESOURCE SOURCE IDENTIFIER Oligomycin A Chemscene Cat#CS-0007502 Omigapil Diverchim SA Cat#DIV00075 Ouabain (Acocantherin) Tocris Cat#1076 Rapamycin (Sirolimus) MedChemExpress Cat#HY-10219 Rheb inhibitor NR-1 WuXi, WuHan China N/A Rotenone TRC Canada Cat#R700580 TFB-TBOA Tocris Cat#2532 Thapsigargin Sigma-Aldrich Cat#T9033 UCPH-101 Sigma-Aldrich Cat#BL3H160B7EB4 Critical commercial assays BsaI-HF v2 NEB Cat#R3733 BsmBI-v2 NEB CatR0739 Pierce BCA Protein Assay Kit Thermo Scientific Inc. Cat#23225 GenElute Mammalian Genomic DNA Miniprep Kit Sigma-Aldrich Cat#G1N350 HisPur Ni-NTA Superflow Agarose Thermo Scientific Inc. Cat#25217 KOD Hot Start Master Mix Sigma-Aldrich Cat#71842 Deposited data SF-iGluSnFR-S72A This paper PDB: 8OVN https://www.rcsb.org/structure/8OVN SF-iGluSnFR-S72A in complex with L-aspartate This paper PDB: 8OVO https://www.rcsb.org/structure/8OVO AspSnFR in complex with L-aspartate This paper PDB: 8OVP https://www.rcsb.org/structure/8OVP Mass spectrometry HCT116SLC25A12-KOOE-AspSnFR This paper MetaboLights: MTBLS10035 https://www.ebi.ac.uk/metabolights/ MTBLS10035 Illumina sequencing data This paper ENA: PRJEB75325 https://www.ebi.ac.uk/ ena/browser/view/PRJEB75325 Transcriptomics (SLC expression) RESOLUTE consortium https://re-solute.eu/knowledgebase/gene ENA: SAMN11893676, SAMN11893683, SAMN11893690 Proteomics (SLC expression) RESOLUTE consortium https://re-solute.eu/knowledgebase/gene ProteomeXchange: PXD051747 Metabolomics (GFP-OE) RESOLUTE consortium https://re-solute.eu/resources/ dashboards/metabolomics/: MetaboLights: MTBLS10077 Experimental models: Cell lines HCT116-SLC25A12-KOOE-AspSnFR This paper N/A HEK293-JI-AspSnFR This paper N/A HEK293-JI-SLC1A3 RESOLUTE consortium RESOLUTE Identifier: CE029W-F https://re-solute.eu/files/ cellline_reports/CE029W-F.pdf Jump In T-REx HEK 293 Kit Thermo Fisher Scientific Inc. Cat#A15008 Oligonculeotides Listed in Table S5 N/A N/A Recombinant DNA pcDNA3.1/cyto-AspSnFr Neomycin This paper Addgene #201394 pcDNA3.1/cyto-AspSnFR Zeocin This paper N/A pET51b(+)/AspSnFR This paper N/A pET51b(+)/AspSnFR lacking mScarlet-I This paper N/A pJTI/cyto-AspSnFR This paper N/A pJTI/R4 Int, Thermo Fisher Scientific Inc. Cat#A15008 pSF-EF1a/EBFP-gpASNase1 This paper N/A (Continued on next page) Cell Chemical Biology 31, 1529–1541.e1–e12, August 15, 2024 e2 .. REAGENT or RESOURCE SOURCE IDENTIFIER pSF-EF1a/gpASNase1 This paper N/A DMS Library, Site-Saturation Variant Library Twist Bioscience N/A Software and algorithms BBtools https://jgi.doe.gov/data-and-tools/ software-tools/bbtools/ https://sourceforge.net/projects/bbmap/ CellProfiler Image analysis Software (OpenSource, Python based) Broad Institute of MIT and Harvard https://cellprofiler.org ChemDraw Professional 22.0 PerkinElmer https://perkinelmer.com FIJI/ImageJ Image processing package (OpenSource, Java based) National Institutes of Health https://imagej.net/ GraphPad Prism 9 GraphPad https://graphpad.com Je demultiplex gbcs.embl.de/Je https://github.com/gbcs-embl/Je JIPipe 1.81 Hans Knöll Institute (HKI), Jena, Germany https://jipipe.hki-jena.de/ Mutagenesis-visualization https://mutagenesis-visualization. readthedocs.io/en/latest/ https://github.com/fhidalgor/ mutagenesis_visualization Other m-Slide 18 Well Ibiditreat Ibidi Cat#81816 m-Slide VI 0.4 Ibidi Cat#80604 384-well plates Corning Cat#3573 96-well plates PerkinElmer Cat#6055260 96-well plates, cell-grade, black, transparent F-bottom BRANDplus Cat#735-2104 Amicon Ultra 4 mL Centrifugal Filters Merck Cat#UFC803024 (30 kDa) Amicon Ultra 4 mL Centrifugal Filters Merck Cat#UFC805024 (50 kDa) Aquamax DW4 microplate washer Molecular Devices LLC.
Transcriptomics:Article Title: AspSnFR: A genetically encoded biosensor for real-time monitoring of aspartate in live cells.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Donkey anti-Goat IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 1mg Thermo Fisher Scientific Inc. Cat#A32849; RRID: AB_2762840 Goat pAb anti HA-tag antibody Novus Bio Cat#NB600-362; RRID: AB_10124937 Bacterial and virus strains BL21(DE3)-pLysS Millipore Cat#69451-3 E. Cloni 10G Lucigen Cat#60117-1 XL1-Blue Agilent Cat#200249 Chemicals, peptides, and recombinant proteins 21 L-amino acids Sigma-Aldrich Cat#LAA21 Ampicillin Sigma-Aldrich Cat#A9393 Antimycin A1 Sigma-Aldrich Cat#A8674 AOA 2-(Aminooxy)Acetic Acid Sigma-Aldrich Cat#C13408 ASCT2 inhibitor WuXi, Wuhan, China N/A BMS-303141 Medchemexpress Cat#HY-16107 Bumetanide Sigma-Aldrich Cat#B3023 Cariporide Sigma-Aldrich Cat#SML1360 CB-839 (Telaglenastat) Medchemexpress Cat#HY-12248 CCCP Sigma-Aldrich Cat#C2759 Cefatriaxone Medchemexpress Cat#HYB-0712B CellLytic M Sigma-Aldrich Cat#C2978 Chloroquine Enamine Cat#EN300-120683 Chlorothiazide ComGenex Ltd, CM-Technology, Budapest, Hungary N/A Cidofovir Waterstone Cat#40997 Compound 968 (GlutaminaseC-IN-1) Sigma-Aldrich Cat#SML1327 Cytochalasin B Cayman Europe Cat#11328 Daporinad (FK866) MedChemExpress Cat#HY-50876 D-aspartate Carl Roth Cat#7873.2 Doxycycline hyclate Sigma-Aldrich Cat#D9891 Furosemide (Desdemin) Enamine Cat#EN300-126738 Gibson assembly mix Home made N/A HCS Cellmask DeepRed Thermo Fisher Scientific Inc. Cat#32721 Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in Water Thermo Fisher Scientific Inc. Cat#H3510 Hydroxychloroquine ABCR Cat#AB564120 Ivosidenib (Tibsovo) MedKoo Biosciences Cat#206148 L-Aspartic Acid (Asp) MerckSigmaAldrich Cat#A9256 Lc-BPE Aurigene, Karnataka 560100, India N/A L-Glutamic Acid (Glu) Sigma-Aldrich Cat#G1251 L-Glutamine (Gln) Sigma-Aldrich Cat#G3126 Losartan ABCR Cat#AB489654 Metformin Sigma-Aldrich Cat#D5035 MitoQ Sigma-Aldrich Cat#ATE959438891 Mitotracker Orange Thermo Fisher Scientific Inc. Cat#M7510 NMN(+) Fluka Cat#72342 (Continued on next page) e1 Cell Chemical Biology 31, 1529–1541.e1–e12, August 15, 2024 .. REAGENT or RESOURCE SOURCE IDENTIFIER Oligomycin A Chemscene Cat#CS-0007502 Omigapil Diverchim SA Cat#DIV00075 Ouabain (Acocantherin) Tocris Cat#1076 Rapamycin (Sirolimus) MedChemExpress Cat#HY-10219 Rheb inhibitor NR-1 WuXi, WuHan China N/A Rotenone TRC Canada Cat#R700580 TFB-TBOA Tocris Cat#2532 Thapsigargin Sigma-Aldrich Cat#T9033 UCPH-101 Sigma-Aldrich Cat#BL3H160B7EB4 Critical commercial assays BsaI-HF v2 NEB Cat#R3733 BsmBI-v2 NEB CatR0739 Pierce BCA Protein Assay Kit Thermo Scientific Inc. Cat#23225 GenElute Mammalian Genomic DNA Miniprep Kit Sigma-Aldrich Cat#G1N350 HisPur Ni-NTA Superflow Agarose Thermo Scientific Inc. Cat#25217 KOD Hot Start Master Mix Sigma-Aldrich Cat#71842 Deposited data SF-iGluSnFR-S72A This paper PDB: 8OVN https://www.rcsb.org/structure/8OVN SF-iGluSnFR-S72A in complex with L-aspartate This paper PDB: 8OVO https://www.rcsb.org/structure/8OVO AspSnFR in complex with L-aspartate This paper PDB: 8OVP https://www.rcsb.org/structure/8OVP Mass spectrometry HCT116SLC25A12-KOOE-AspSnFR This paper MetaboLights: MTBLS10035 https://www.ebi.ac.uk/metabolights/ MTBLS10035 Illumina sequencing data This paper ENA: PRJEB75325 https://www.ebi.ac.uk/ ena/browser/view/PRJEB75325 Transcriptomics (SLC expression) RESOLUTE consortium https://re-solute.eu/knowledgebase/gene ENA: SAMN11893676, SAMN11893683, SAMN11893690 Proteomics (SLC expression) RESOLUTE consortium https://re-solute.eu/knowledgebase/gene ProteomeXchange: PXD051747 Metabolomics (GFP-OE) RESOLUTE consortium https://re-solute.eu/resources/ dashboards/metabolomics/: MetaboLights: MTBLS10077 Experimental models: Cell lines HCT116-SLC25A12-KOOE-AspSnFR This paper N/A HEK293-JI-AspSnFR This paper N/A HEK293-JI-SLC1A3 RESOLUTE consortium RESOLUTE Identifier: CE029W-F https://re-solute.eu/files/ cellline_reports/CE029W-F.pdf Jump In T-REx HEK 293 Kit Thermo Fisher Scientific Inc. Cat#A15008 Oligonculeotides Listed in Table S5 N/A N/A Recombinant DNA pcDNA3.1/cyto-AspSnFr Neomycin This paper Addgene #201394 pcDNA3.1/cyto-AspSnFR Zeocin This paper N/A pET51b(+)/AspSnFR This paper N/A pET51b(+)/AspSnFR lacking mScarlet-I This paper N/A pJTI/cyto-AspSnFR This paper N/A pJTI/R4 Int, Thermo Fisher Scientific Inc. Cat#A15008 pSF-EF1a/EBFP-gpASNase1 This paper N/A (Continued on next page) Cell Chemical Biology 31, 1529–1541.e1–e12, August 15, 2024 e2 .. REAGENT or RESOURCE SOURCE IDENTIFIER pSF-EF1a/gpASNase1 This paper N/A DMS Library, Site-Saturation Variant Library Twist Bioscience N/A Software and algorithms BBtools https://jgi.doe.gov/data-and-tools/ software-tools/bbtools/ https://sourceforge.net/projects/bbmap/ CellProfiler Image analysis Software (OpenSource, Python based) Broad Institute of MIT and Harvard https://cellprofiler.org ChemDraw Professional 22.0 PerkinElmer https://perkinelmer.com FIJI/ImageJ Image processing package (OpenSource, Java based) National Institutes of Health https://imagej.net/ GraphPad Prism 9 GraphPad https://graphpad.com Je demultiplex gbcs.embl.de/Je https://github.com/gbcs-embl/Je JIPipe 1.81 Hans Knöll Institute (HKI), Jena, Germany https://jipipe.hki-jena.de/ Mutagenesis-visualization https://mutagenesis-visualization. readthedocs.io/en/latest/ https://github.com/fhidalgor/ mutagenesis_visualization Other m-Slide 18 Well Ibiditreat Ibidi Cat#81816 m-Slide VI 0.4 Ibidi Cat#80604 384-well plates Corning Cat#3573 96-well plates PerkinElmer Cat#6055260 96-well plates, cell-grade, black, transparent F-bottom BRANDplus Cat#735-2104 Amicon Ultra 4 mL Centrifugal Filters Merck Cat#UFC803024 (30 kDa) Amicon Ultra 4 mL Centrifugal Filters Merck Cat#UFC805024 (50 kDa) Aquamax DW4 microplate washer Molecular Devices LLC.
Expressing:Article Title: AspSnFR: A genetically encoded biosensor for real-time monitoring of aspartate in live cells.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Donkey anti-Goat IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 1mg Thermo Fisher Scientific Inc. Cat#A32849; RRID: AB_2762840 Goat pAb anti HA-tag antibody Novus Bio Cat#NB600-362; RRID: AB_10124937 Bacterial and virus strains BL21(DE3)-pLysS Millipore Cat#69451-3 E. Cloni 10G Lucigen Cat#60117-1 XL1-Blue Agilent Cat#200249 Chemicals, peptides, and recombinant proteins 21 L-amino acids Sigma-Aldrich Cat#LAA21 Ampicillin Sigma-Aldrich Cat#A9393 Antimycin A1 Sigma-Aldrich Cat#A8674 AOA 2-(Aminooxy)Acetic Acid Sigma-Aldrich Cat#C13408 ASCT2 inhibitor WuXi, Wuhan, China N/A BMS-303141 Medchemexpress Cat#HY-16107 Bumetanide Sigma-Aldrich Cat#B3023 Cariporide Sigma-Aldrich Cat#SML1360 CB-839 (Telaglenastat) Medchemexpress Cat#HY-12248 CCCP Sigma-Aldrich Cat#C2759 Cefatriaxone Medchemexpress Cat#HYB-0712B CellLytic M Sigma-Aldrich Cat#C2978 Chloroquine Enamine Cat#EN300-120683 Chlorothiazide ComGenex Ltd, CM-Technology, Budapest, Hungary N/A Cidofovir Waterstone Cat#40997 Compound 968 (GlutaminaseC-IN-1) Sigma-Aldrich Cat#SML1327 Cytochalasin B Cayman Europe Cat#11328 Daporinad (FK866) MedChemExpress Cat#HY-50876 D-aspartate Carl Roth Cat#7873.2 Doxycycline hyclate Sigma-Aldrich Cat#D9891 Furosemide (Desdemin) Enamine Cat#EN300-126738 Gibson assembly mix Home made N/A HCS Cellmask DeepRed Thermo Fisher Scientific Inc. Cat#32721 Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in Water Thermo Fisher Scientific Inc. Cat#H3510 Hydroxychloroquine ABCR Cat#AB564120 Ivosidenib (Tibsovo) MedKoo Biosciences Cat#206148 L-Aspartic Acid (Asp) MerckSigmaAldrich Cat#A9256 Lc-BPE Aurigene, Karnataka 560100, India N/A L-Glutamic Acid (Glu) Sigma-Aldrich Cat#G1251 L-Glutamine (Gln) Sigma-Aldrich Cat#G3126 Losartan ABCR Cat#AB489654 Metformin Sigma-Aldrich Cat#D5035 MitoQ Sigma-Aldrich Cat#ATE959438891 Mitotracker Orange Thermo Fisher Scientific Inc. Cat#M7510 NMN(+) Fluka Cat#72342 (Continued on next page) e1 Cell Chemical Biology 31, 1529–1541.e1–e12, August 15, 2024 .. REAGENT or RESOURCE SOURCE IDENTIFIER Oligomycin A Chemscene Cat#CS-0007502 Omigapil Diverchim SA Cat#DIV00075 Ouabain (Acocantherin) Tocris Cat#1076 Rapamycin (Sirolimus) MedChemExpress Cat#HY-10219 Rheb inhibitor NR-1 WuXi, WuHan China N/A Rotenone TRC Canada Cat#R700580 TFB-TBOA Tocris Cat#2532 Thapsigargin Sigma-Aldrich Cat#T9033 UCPH-101 Sigma-Aldrich Cat#BL3H160B7EB4 Critical commercial assays BsaI-HF v2 NEB Cat#R3733 BsmBI-v2 NEB CatR0739 Pierce BCA Protein Assay Kit Thermo Scientific Inc. Cat#23225 GenElute Mammalian Genomic DNA Miniprep Kit Sigma-Aldrich Cat#G1N350 HisPur Ni-NTA Superflow Agarose Thermo Scientific Inc. Cat#25217 KOD Hot Start Master Mix Sigma-Aldrich Cat#71842 Deposited data SF-iGluSnFR-S72A This paper PDB: 8OVN https://www.rcsb.org/structure/8OVN SF-iGluSnFR-S72A in complex with L-aspartate This paper PDB: 8OVO https://www.rcsb.org/structure/8OVO AspSnFR in complex with L-aspartate This paper PDB: 8OVP https://www.rcsb.org/structure/8OVP Mass spectrometry HCT116SLC25A12-KOOE-AspSnFR This paper MetaboLights: MTBLS10035 https://www.ebi.ac.uk/metabolights/ MTBLS10035 Illumina sequencing data This paper ENA: PRJEB75325 https://www.ebi.ac.uk/ ena/browser/view/PRJEB75325 Transcriptomics (SLC expression) RESOLUTE consortium https://re-solute.eu/knowledgebase/gene ENA: SAMN11893676, SAMN11893683, SAMN11893690 Proteomics (SLC expression) RESOLUTE consortium https://re-solute.eu/knowledgebase/gene ProteomeXchange: PXD051747 Metabolomics (GFP-OE) RESOLUTE consortium https://re-solute.eu/resources/ dashboards/metabolomics/: MetaboLights: MTBLS10077 Experimental models: Cell lines HCT116-SLC25A12-KOOE-AspSnFR This paper N/A HEK293-JI-AspSnFR This paper N/A HEK293-JI-SLC1A3 RESOLUTE consortium RESOLUTE Identifier: CE029W-F https://re-solute.eu/files/ cellline_reports/CE029W-F.pdf Jump In T-REx HEK 293 Kit Thermo Fisher Scientific Inc. Cat#A15008 Oligonculeotides Listed in Table S5 N/A N/A Recombinant DNA pcDNA3.1/cyto-AspSnFr Neomycin This paper Addgene #201394 pcDNA3.1/cyto-AspSnFR Zeocin This paper N/A pET51b(+)/AspSnFR This paper N/A pET51b(+)/AspSnFR lacking mScarlet-I This paper N/A pJTI/cyto-AspSnFR This paper N/A pJTI/R4 Int, Thermo Fisher Scientific Inc. Cat#A15008 pSF-EF1a/EBFP-gpASNase1 This paper N/A (Continued on next page) Cell Chemical Biology 31, 1529–1541.e1–e12, August 15, 2024 e2 .. REAGENT or RESOURCE SOURCE IDENTIFIER pSF-EF1a/gpASNase1 This paper N/A DMS Library, Site-Saturation Variant Library Twist Bioscience N/A Software and algorithms BBtools https://jgi.doe.gov/data-and-tools/ software-tools/bbtools/ https://sourceforge.net/projects/bbmap/ CellProfiler Image analysis Software (OpenSource, Python based) Broad Institute of MIT and Harvard https://cellprofiler.org ChemDraw Professional 22.0 PerkinElmer https://perkinelmer.com FIJI/ImageJ Image processing package (OpenSource, Java based) National Institutes of Health https://imagej.net/ GraphPad Prism 9 GraphPad https://graphpad.com Je demultiplex gbcs.embl.de/Je https://github.com/gbcs-embl/Je JIPipe 1.81 Hans Knöll Institute (HKI), Jena, Germany https://jipipe.hki-jena.de/ Mutagenesis-visualization https://mutagenesis-visualization. readthedocs.io/en/latest/ https://github.com/fhidalgor/ mutagenesis_visualization Other m-Slide 18 Well Ibiditreat Ibidi Cat#81816 m-Slide VI 0.4 Ibidi Cat#80604 384-well plates Corning Cat#3573 96-well plates PerkinElmer Cat#6055260 96-well plates, cell-grade, black, transparent F-bottom BRANDplus Cat#735-2104 Amicon Ultra 4 mL Centrifugal Filters Merck Cat#UFC803024 (30 kDa) Amicon Ultra 4 mL Centrifugal Filters Merck Cat#UFC805024 (50 kDa) Aquamax DW4 microplate washer Molecular Devices LLC.
Synthesized:Article Title: Scalable TCR synthesis and screening enable antigen reactivity mapping in vitiligo.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies APC anti-human CD69 (clone FN50) Biolegend Cat#310910; RRID: AB_314845 PE anti-human CD19 (clone HIB19) Biolegend Cat#302208; RRID: AB_314238 APC anti-human TCR α/β (clone IP26) Biolegend Cat#306718; RRID: AB_10612569 PE anti-human TCR α/β (clone IP26) Biolegend Cat#306708; RRID: AB_314646 Bacterial and virus strains Stbl3 chemical competent cells ThermoFisher Cat#C737303 ElectroMAX® DH10β electrocompetent E. coli ThermoFisher Cat#18290015 Biological samples Suction blister biopsies Vitiligo Clinic and Research Center, University of Massachusetts Chan Medical School N/A Chemicals, peptides, and recombinant proteins Synthesized peptides Genscript Table S2 Phorbol 12-myristate 13-acetate (PMA) ThermoFisher Cat#J63916.MA DEAE-dextran hydrochloride Sigma Cat#D9885-10G Ionomycin, 96% ThermoFisher Cat#J62448.MCR Critical commercial assays NEBNext Ultra II Q5 Master Mix NEB Cat#M0544L KAPA HiFi HotStart Readymix Roche Cat# KK2601 NEBridge Ligase Master Mix NEB Cat#M1100 NEBridge Golden Gate Assembly Kit (BsmBI-v2) NEB CatE1602 NEBridge Golden Gate Assembly Kit (BsaI-HF-v2) NEB Cat#E1601 BsaI-HF-v2 NEB Cat#R3733 BsmBI-v2 NEB Cat#R0739 BbsI-HF NEB Cat#R3539 SapI NEB Cat#R0569 FastDigest XbaI ThermoFisher Cat#FD0685 FastDigest Buffer ThermoFisher Cat#B64 Nuclease-free water (not DEPC-treated) ThermoFisher Cat#AM9937 Shrimp alkaline phosphatase (rSAP) NEB Cat#M0371 ElectroMAX DH10B E. coli ThermoFisher Cat#18290015 MCE membrane filter, 0.025 μm pore size Millipore Sigma Cat#VSWP02500 Chloramphenicol Millipore Sigma Cat#C0378 Electroporation cuvette (1 mm gap) VWR Cat#89047-206 SPRIselect beads Beckman Coulter Cat#B23317 GeneJET Plasmid Miniprep Kit ThermoFisher Cat#K0502 GeneJET Gel Extraction Kit ThermoFisher Cat#K0692 TransIT®-Lenti Transfection Reagent Millipore Sigma Cat#MIR6603 Quikchange Lightning Site-Directed Mutagenesis Kit Agilent Cat#210518 Genscript Genbuilder Gibson Mix Genscript Cat#L00701-50 Ligation sequencing DNA V14 Oxford Nanopore Technologies Cat#SQK-LSK114 Chromium GEM-X Single Cell 5 ′ Kit v3 10X Genomics Cat#PN-1000695 Chromium Next GEM Single Cell 5 ′ Kit v2 10X Genomics Cat#PN-1000263 (Continued on next page) e1 Immunity 59, 1–17.e1–e9, February 10, 2026 .. HEK293T cells (ATCC CRL-11268) were cultured in DMEM (ATCC) supplemented with 10% fetal bovine serum (FBS; Atlanta biologics) and penicillin-streptomycin (Gibco).
Article Title: An AI-driven workflow for the accelerated optimization of cell-free protein synthesis
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-6xHistidin-tag Sigma Cat#SAB2702220; RRID: AB_3095553 anti-GFP-tag Cell signaling Cat#2555; RRID: AB_10692764 anti-HiBiT-tag Promega Cat#N720A Bacterial and virus strains Escherichia coli DH5α Lab stock Escherichia coli BL21 (DE3) Lab stock Escherichia coli W3110 Lab stock Biological samples E. coli cell-free lysates Synthelis Biotech HeLa Lysate ThermoScientific Cat#88882 Chemicals, peptides, and recombinant proteins HEPES Sigma-Aldrich Cat#H3375 Nucleotide mix - Adenosine 5-triphosphate dipotassium salt hydrate Sigma-Aldrich Cat#A8937 Nucleotide mix - Guanosine 5ʹ-Triphosphate, Disodium Salt Sigma-Aldrich Cat#371701 Nucleotide mix - Sel disodique cytidine-5 ′ -triphosphate, 98+ % Thermo Scientific Chemicals Cat#J62238.ME Nucleotide mix - Uridine 5 ′ -triphosphate, sel trisodique, hydraté , 90% Thermo Scientific Chemicals Cat#226310010 3- PGA (Acide D-(− )-3-phosphoglycé rique disodium salt) Sigma-Aldrich Cat#P8877 Spermidine Sigma-Aldrich Cat#85558 Creatine phosphate crystalline, sup à 97% Sigma-Aldrich Cat#10621714001 PEG 8000 Sigma-Aldrich Cat#89510 Mg-glutamate Sigma-Aldrich Cat#49605 K-glutamate Alfa Aesar Cat#A17232 Amino acids - L-Alanine Sigma Cat#A7627 Amino acids - L-Arginine Sigma Cat#A5006 Amino acids - Glycine Sigma Cat#G7126 Amino acids - L-Histidine Merck Cat#1.04351.0100 Amino acids - L-Lysine Sigma Cat#L5626 Amino acids - L-Proline Sigma Cat#P0380 Amino acids - L-Serine Sigma Cat#S4500 Amino acids - L-Threonine Sigma Cat#T8625 Amino acids - L-Valine Sigma Cat#V0500 Amino acids - L-Aspartic Acid Sigma Cat#A9256 Amino acids - L-Asparagine anhydrous Sigma Cat#A0884 Amino acids - L-Cystine Sigma Cat#C8755 Amino acids - L-Glutamine Sigma Cat#G3126 Amino acids - L-Leucine Sigma Cat#L8000 Amino acids - L-Methionine Sigma Cat#64319 Amino acids - L-Tryptophan Sigma Cat#T8941 (Continued on next page) iScience 28, 113599, October 17, 2025 e1 .. REAGENT or RESOURCE SOURCE IDENTIFIER Amino acids - L-Tyrosine Sigma Cat#T3754 Amino acids - L-Phenylalanine Sigma Cat#78019 Amino acids - lsoleucine Sigma Cat#I7403 BsmBI-v2 NEB CatR0739S BbsI NEB Cat#R0539S BsaI NEB Cat#R3733S DpnI NEB Cat#R0176L BamHI Promega Cat#R6021 EcoRV Promega Cat#R6351 T4 DNA ligase Promega Cat#M1801 T4 DNA ligase NEB Cat#M0202 Ampicillin Sigma–Aldrich Cat#A1593 (IPTG) isopropyl -d-1-thiogalactopyranoside Sigma–Aldrich Cat#I5502 Critical commercial assays Q5 High-Fidelity 2X Master Mix NEB Cat#M0492L PCR purification kit NEB Cat#T1130 NucleoBond Xtra Maxi kit Macherey-Nagel Cat#740414.1 Nano-Glo® HiBiT Lytic Detection System Promega Cat#N3030 Nano-Glo® HiBiT Lytic Detection System Promega Cat#N3040 Deposited data icfree-ml source code https://github.com/brsynth/icfree-ml https://doi.org/10.5281/zenodo.14900104 icfree-ml conda packages https://anaconda.org/bioconda/icfree-ml https://doi.org/10.5281/zenodo.14893904 icfree-ml Galaxy wrappers https://toolshed.g2.bx.psu.edu https://doi.org/10.5281/zenodo.14893963 AI-CellFree - Init Galaxy workflow https://galaxy-synbiocad.org/ u/joan/w/icfree-init https://doi.org/10.5281/zenodo.14901023 AI-CellFree - Core Galaxy workflow https://galaxy-synbiocad.org/ u/joan/w/icfree-core https://doi.org/10.5281/zenodo.14899706 Experimental models: Organisms/strains Escherichia coli DH5α Escherichia coli BL21 (DE3) Escherichia coli W3110 Oligonucleotides See Table S3 Recombinant DNA Colicin M Synthesized by GeneCust Colicin E1 Synthesized by GeneCust GFP1-10 Synthesized by GeneCust GFP11 Synthesized by GeneCust His-GFP1-10 This study His-GFP11-Colicin M This study His-GFP11-Colicin E1 This study His-HiBiT-Colicin M This study His-HiBiT-Colicin E1 This study His-HiBiT-sfGFP This study Software and algorithms Echo Cherry Pick software Beckman Coulter Protocol Designer Opentrons Image Lab TM Software Bio-Rad Laboratories ChatGPT-4 Openai e2 iScience 28, 113599, October 17, 2025 ..
Membrane:Article Title: Scalable TCR synthesis and screening enable antigen reactivity mapping in vitiligo.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies APC anti-human CD69 (clone FN50) Biolegend Cat#310910; RRID: AB_314845 PE anti-human CD19 (clone HIB19) Biolegend Cat#302208; RRID: AB_314238 APC anti-human TCR α/β (clone IP26) Biolegend Cat#306718; RRID: AB_10612569 PE anti-human TCR α/β (clone IP26) Biolegend Cat#306708; RRID: AB_314646 Bacterial and virus strains Stbl3 chemical competent cells ThermoFisher Cat#C737303 ElectroMAX® DH10β electrocompetent E. coli ThermoFisher Cat#18290015 Biological samples Suction blister biopsies Vitiligo Clinic and Research Center, University of Massachusetts Chan Medical School N/A Chemicals, peptides, and recombinant proteins Synthesized peptides Genscript Table S2 Phorbol 12-myristate 13-acetate (PMA) ThermoFisher Cat#J63916.MA DEAE-dextran hydrochloride Sigma Cat#D9885-10G Ionomycin, 96% ThermoFisher Cat#J62448.MCR Critical commercial assays NEBNext Ultra II Q5 Master Mix NEB Cat#M0544L KAPA HiFi HotStart Readymix Roche Cat# KK2601 NEBridge Ligase Master Mix NEB Cat#M1100 NEBridge Golden Gate Assembly Kit (BsmBI-v2) NEB CatE1602 NEBridge Golden Gate Assembly Kit (BsaI-HF-v2) NEB Cat#E1601 BsaI-HF-v2 NEB Cat#R3733 BsmBI-v2 NEB Cat#R0739 BbsI-HF NEB Cat#R3539 SapI NEB Cat#R0569 FastDigest XbaI ThermoFisher Cat#FD0685 FastDigest Buffer ThermoFisher Cat#B64 Nuclease-free water (not DEPC-treated) ThermoFisher Cat#AM9937 Shrimp alkaline phosphatase (rSAP) NEB Cat#M0371 ElectroMAX DH10B E. coli ThermoFisher Cat#18290015 MCE membrane filter, 0.025 μm pore size Millipore Sigma Cat#VSWP02500 Chloramphenicol Millipore Sigma Cat#C0378 Electroporation cuvette (1 mm gap) VWR Cat#89047-206 SPRIselect beads Beckman Coulter Cat#B23317 GeneJET Plasmid Miniprep Kit ThermoFisher Cat#K0502 GeneJET Gel Extraction Kit ThermoFisher Cat#K0692 TransIT®-Lenti Transfection Reagent Millipore Sigma Cat#MIR6603 Quikchange Lightning Site-Directed Mutagenesis Kit Agilent Cat#210518 Genscript Genbuilder Gibson Mix Genscript Cat#L00701-50 Ligation sequencing DNA V14 Oxford Nanopore Technologies Cat#SQK-LSK114 Chromium GEM-X Single Cell 5 ′ Kit v3 10X Genomics Cat#PN-1000695 Chromium Next GEM Single Cell 5 ′ Kit v2 10X Genomics Cat#PN-1000263 (Continued on next page) e1 Immunity 59, 1–17.e1–e9, February 10, 2026 .. HEK293T cells (ATCC CRL-11268) were cultured in DMEM (ATCC) supplemented with 10% fetal bovine serum (FBS; Atlanta biologics) and penicillin-streptomycin (Gibco).
Pore Size:Article Title: Scalable TCR synthesis and screening enable antigen reactivity mapping in vitiligo.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies APC anti-human CD69 (clone FN50) Biolegend Cat#310910; RRID: AB_314845 PE anti-human CD19 (clone HIB19) Biolegend Cat#302208; RRID: AB_314238 APC anti-human TCR α/β (clone IP26) Biolegend Cat#306718; RRID: AB_10612569 PE anti-human TCR α/β (clone IP26) Biolegend Cat#306708; RRID: AB_314646 Bacterial and virus strains Stbl3 chemical competent cells ThermoFisher Cat#C737303 ElectroMAX® DH10β electrocompetent E. coli ThermoFisher Cat#18290015 Biological samples Suction blister biopsies Vitiligo Clinic and Research Center, University of Massachusetts Chan Medical School N/A Chemicals, peptides, and recombinant proteins Synthesized peptides Genscript Table S2 Phorbol 12-myristate 13-acetate (PMA) ThermoFisher Cat#J63916.MA DEAE-dextran hydrochloride Sigma Cat#D9885-10G Ionomycin, 96% ThermoFisher Cat#J62448.MCR Critical commercial assays NEBNext Ultra II Q5 Master Mix NEB Cat#M0544L KAPA HiFi HotStart Readymix Roche Cat# KK2601 NEBridge Ligase Master Mix NEB Cat#M1100 NEBridge Golden Gate Assembly Kit (BsmBI-v2) NEB CatE1602 NEBridge Golden Gate Assembly Kit (BsaI-HF-v2) NEB Cat#E1601 BsaI-HF-v2 NEB Cat#R3733 BsmBI-v2 NEB Cat#R0739 BbsI-HF NEB Cat#R3539 SapI NEB Cat#R0569 FastDigest XbaI ThermoFisher Cat#FD0685 FastDigest Buffer ThermoFisher Cat#B64 Nuclease-free water (not DEPC-treated) ThermoFisher Cat#AM9937 Shrimp alkaline phosphatase (rSAP) NEB Cat#M0371 ElectroMAX DH10B E. coli ThermoFisher Cat#18290015 MCE membrane filter, 0.025 μm pore size Millipore Sigma Cat#VSWP02500 Chloramphenicol Millipore Sigma Cat#C0378 Electroporation cuvette (1 mm gap) VWR Cat#89047-206 SPRIselect beads Beckman Coulter Cat#B23317 GeneJET Plasmid Miniprep Kit ThermoFisher Cat#K0502 GeneJET Gel Extraction Kit ThermoFisher Cat#K0692 TransIT®-Lenti Transfection Reagent Millipore Sigma Cat#MIR6603 Quikchange Lightning Site-Directed Mutagenesis Kit Agilent Cat#210518 Genscript Genbuilder Gibson Mix Genscript Cat#L00701-50 Ligation sequencing DNA V14 Oxford Nanopore Technologies Cat#SQK-LSK114 Chromium GEM-X Single Cell 5 ′ Kit v3 10X Genomics Cat#PN-1000695 Chromium Next GEM Single Cell 5 ′ Kit v2 10X Genomics Cat#PN-1000263 (Continued on next page) e1 Immunity 59, 1–17.e1–e9, February 10, 2026 .. HEK293T cells (ATCC CRL-11268) were cultured in DMEM (ATCC) supplemented with 10% fetal bovine serum (FBS; Atlanta biologics) and penicillin-streptomycin (Gibco).
Electroporation:Article Title: Scalable TCR synthesis and screening enable antigen reactivity mapping in vitiligo.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies APC anti-human CD69 (clone FN50) Biolegend Cat#310910; RRID: AB_314845 PE anti-human CD19 (clone HIB19) Biolegend Cat#302208; RRID: AB_314238 APC anti-human TCR α/β (clone IP26) Biolegend Cat#306718; RRID: AB_10612569 PE anti-human TCR α/β (clone IP26) Biolegend Cat#306708; RRID: AB_314646 Bacterial and virus strains Stbl3 chemical competent cells ThermoFisher Cat#C737303 ElectroMAX® DH10β electrocompetent E. coli ThermoFisher Cat#18290015 Biological samples Suction blister biopsies Vitiligo Clinic and Research Center, University of Massachusetts Chan Medical School N/A Chemicals, peptides, and recombinant proteins Synthesized peptides Genscript Table S2 Phorbol 12-myristate 13-acetate (PMA) ThermoFisher Cat#J63916.MA DEAE-dextran hydrochloride Sigma Cat#D9885-10G Ionomycin, 96% ThermoFisher Cat#J62448.MCR Critical commercial assays NEBNext Ultra II Q5 Master Mix NEB Cat#M0544L KAPA HiFi HotStart Readymix Roche Cat# KK2601 NEBridge Ligase Master Mix NEB Cat#M1100 NEBridge Golden Gate Assembly Kit (BsmBI-v2) NEB CatE1602 NEBridge Golden Gate Assembly Kit (BsaI-HF-v2) NEB Cat#E1601 BsaI-HF-v2 NEB Cat#R3733 BsmBI-v2 NEB Cat#R0739 BbsI-HF NEB Cat#R3539 SapI NEB Cat#R0569 FastDigest XbaI ThermoFisher Cat#FD0685 FastDigest Buffer ThermoFisher Cat#B64 Nuclease-free water (not DEPC-treated) ThermoFisher Cat#AM9937 Shrimp alkaline phosphatase (rSAP) NEB Cat#M0371 ElectroMAX DH10B E. coli ThermoFisher Cat#18290015 MCE membrane filter, 0.025 μm pore size Millipore Sigma Cat#VSWP02500 Chloramphenicol Millipore Sigma Cat#C0378 Electroporation cuvette (1 mm gap) VWR Cat#89047-206 SPRIselect beads Beckman Coulter Cat#B23317 GeneJET Plasmid Miniprep Kit ThermoFisher Cat#K0502 GeneJET Gel Extraction Kit ThermoFisher Cat#K0692 TransIT®-Lenti Transfection Reagent Millipore Sigma Cat#MIR6603 Quikchange Lightning Site-Directed Mutagenesis Kit Agilent Cat#210518 Genscript Genbuilder Gibson Mix Genscript Cat#L00701-50 Ligation sequencing DNA V14 Oxford Nanopore Technologies Cat#SQK-LSK114 Chromium GEM-X Single Cell 5 ′ Kit v3 10X Genomics Cat#PN-1000695 Chromium Next GEM Single Cell 5 ′ Kit v2 10X Genomics Cat#PN-1000263 (Continued on next page) e1 Immunity 59, 1–17.e1–e9, February 10, 2026 .. HEK293T cells (ATCC CRL-11268) were cultured in DMEM (ATCC) supplemented with 10% fetal bovine serum (FBS; Atlanta biologics) and penicillin-streptomycin (Gibco).
Plasmid Preparation:Article Title: Scalable TCR synthesis and screening enable antigen reactivity mapping in vitiligo.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies APC anti-human CD69 (clone FN50) Biolegend Cat#310910; RRID: AB_314845 PE anti-human CD19 (clone HIB19) Biolegend Cat#302208; RRID: AB_314238 APC anti-human TCR α/β (clone IP26) Biolegend Cat#306718; RRID: AB_10612569 PE anti-human TCR α/β (clone IP26) Biolegend Cat#306708; RRID: AB_314646 Bacterial and virus strains Stbl3 chemical competent cells ThermoFisher Cat#C737303 ElectroMAX® DH10β electrocompetent E. coli ThermoFisher Cat#18290015 Biological samples Suction blister biopsies Vitiligo Clinic and Research Center, University of Massachusetts Chan Medical School N/A Chemicals, peptides, and recombinant proteins Synthesized peptides Genscript Table S2 Phorbol 12-myristate 13-acetate (PMA) ThermoFisher Cat#J63916.MA DEAE-dextran hydrochloride Sigma Cat#D9885-10G Ionomycin, 96% ThermoFisher Cat#J62448.MCR Critical commercial assays NEBNext Ultra II Q5 Master Mix NEB Cat#M0544L KAPA HiFi HotStart Readymix Roche Cat# KK2601 NEBridge Ligase Master Mix NEB Cat#M1100 NEBridge Golden Gate Assembly Kit (BsmBI-v2) NEB CatE1602 NEBridge Golden Gate Assembly Kit (BsaI-HF-v2) NEB Cat#E1601 BsaI-HF-v2 NEB Cat#R3733 BsmBI-v2 NEB Cat#R0739 BbsI-HF NEB Cat#R3539 SapI NEB Cat#R0569 FastDigest XbaI ThermoFisher Cat#FD0685 FastDigest Buffer ThermoFisher Cat#B64 Nuclease-free water (not DEPC-treated) ThermoFisher Cat#AM9937 Shrimp alkaline phosphatase (rSAP) NEB Cat#M0371 ElectroMAX DH10B E. coli ThermoFisher Cat#18290015 MCE membrane filter, 0.025 μm pore size Millipore Sigma Cat#VSWP02500 Chloramphenicol Millipore Sigma Cat#C0378 Electroporation cuvette (1 mm gap) VWR Cat#89047-206 SPRIselect beads Beckman Coulter Cat#B23317 GeneJET Plasmid Miniprep Kit ThermoFisher Cat#K0502 GeneJET Gel Extraction Kit ThermoFisher Cat#K0692 TransIT®-Lenti Transfection Reagent Millipore Sigma Cat#MIR6603 Quikchange Lightning Site-Directed Mutagenesis Kit Agilent Cat#210518 Genscript Genbuilder Gibson Mix Genscript Cat#L00701-50 Ligation sequencing DNA V14 Oxford Nanopore Technologies Cat#SQK-LSK114 Chromium GEM-X Single Cell 5 ′ Kit v3 10X Genomics Cat#PN-1000695 Chromium Next GEM Single Cell 5 ′ Kit v2 10X Genomics Cat#PN-1000263 (Continued on next page) e1 Immunity 59, 1–17.e1–e9, February 10, 2026 .. HEK293T cells (ATCC CRL-11268) were cultured in DMEM (ATCC) supplemented with 10% fetal bovine serum (FBS; Atlanta biologics) and penicillin-streptomycin (Gibco).
Article Title: Protocol to implement saturation mutagenesis-reinforced functional assays to resolve small-sized variants in disease-related genes.
Article Snippet: .. Continued REAGENT or RESOURCE SOURCE IDENTIFIER Viobility 405/452 fixable dye Miltenyi Biotec Cat#130-130-420 MACS BSA stock solution Miltenyi Biotec Cat#130-091-376 autoMACS rinsing solution Miltenyi Biotec Cat#130-091-222 Human BD Fc Block BD Pharmingen Cat#564220 Q5 high-fidelity DNA polymerase NEB Cat#M0491SVIAL Q5 reaction buffer NEB Cat#B9027SVIAL Q5 High GC Enhancer NEB Cat#B9028AVIAL dNTPs NEB Cat#N0447 BsmBI-v2 NEB CatR0739S BsaI-HFv2 NEB Cat#R3733S XbaI NEB Cat#R0145S BamHI-HF NEB Cat#R3136S Type2M enzyme MspJI NEB Cat#R0661S Type2M enzyme DpnI NEB Cat#R0176S Polybrene Sigma-Aldrich Cat#107689 Critical commercial assays Synthetic single guide RNA kit Synthego N/A NucleoSpin gel and PCR clean-up kit Takara Cat#740609 PureLink midiprep kit Invitrogen Cat#K210014 PureLink genomic DNA mini kit Invitrogen Cat#K182002 SE Cell Line 4D-Nucleofector X Kit S Lonza Cat#V4XC-1032 Experimental models: Cell lines HAP1 Horizon Discovery C631 HEK293T ATCC CRL-3216 MB135 Dr. Stephen J. Tapscott N/A Recombinant DNA Lenti-UbC-FKRP-EF1a-BSD Ma et al. 1 Addgene Plasmid #205150 psPAX2 Dr. Didier Trono Addgene Plasmid #12260 pMD2.G Dr. Didier Trono Addgene Plasmid #12259 Software and algorithms R R Core Team https://www.r-project.org/contributors.html Scripts for oligo design, pre-SMuRF experiments Ma et al. 1 https://doi.org/10.5281/zenodo.13328292 Raw NGS data analytical pipeline Ma et al. 1 https://doi.org/10.5281/zenodo.13328302 Scripts for generating SMuRF scores and downstream analyses Ma et al. 1 https://doi.org/10.5281/zenodo.13328286 Other 0.45 μm PES filter Thermo Scientific Cat#165-0045 40 μm cell strainer Falcon Cat#352340 3D Rocker Benchmark Scientific Cat#B3D1008 Nikon microscope object marker Fisher Scientific Cat#NIKON-MBW10020 10 3 10 mm PYREX cloning cylinders Avantor/VWR Cat#22877-256 TC20 automated cell counter Bio-Rad Cat#1450102 Qubit fluorometer Thermo Scientific Cat#Q33226 STAR Protocols 6, 103909, September 19, 2025 3 Note: Synthego Synthetic Single Guide RNA (sgRNA) Kits can be utilized for sgRNA design and synthesis. ..
Gel Extraction:Article Title: Scalable TCR synthesis and screening enable antigen reactivity mapping in vitiligo.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies APC anti-human CD69 (clone FN50) Biolegend Cat#310910; RRID: AB_314845 PE anti-human CD19 (clone HIB19) Biolegend Cat#302208; RRID: AB_314238 APC anti-human TCR α/β (clone IP26) Biolegend Cat#306718; RRID: AB_10612569 PE anti-human TCR α/β (clone IP26) Biolegend Cat#306708; RRID: AB_314646 Bacterial and virus strains Stbl3 chemical competent cells ThermoFisher Cat#C737303 ElectroMAX® DH10β electrocompetent E. coli ThermoFisher Cat#18290015 Biological samples Suction blister biopsies Vitiligo Clinic and Research Center, University of Massachusetts Chan Medical School N/A Chemicals, peptides, and recombinant proteins Synthesized peptides Genscript Table S2 Phorbol 12-myristate 13-acetate (PMA) ThermoFisher Cat#J63916.MA DEAE-dextran hydrochloride Sigma Cat#D9885-10G Ionomycin, 96% ThermoFisher Cat#J62448.MCR Critical commercial assays NEBNext Ultra II Q5 Master Mix NEB Cat#M0544L KAPA HiFi HotStart Readymix Roche Cat# KK2601 NEBridge Ligase Master Mix NEB Cat#M1100 NEBridge Golden Gate Assembly Kit (BsmBI-v2) NEB CatE1602 NEBridge Golden Gate Assembly Kit (BsaI-HF-v2) NEB Cat#E1601 BsaI-HF-v2 NEB Cat#R3733 BsmBI-v2 NEB Cat#R0739 BbsI-HF NEB Cat#R3539 SapI NEB Cat#R0569 FastDigest XbaI ThermoFisher Cat#FD0685 FastDigest Buffer ThermoFisher Cat#B64 Nuclease-free water (not DEPC-treated) ThermoFisher Cat#AM9937 Shrimp alkaline phosphatase (rSAP) NEB Cat#M0371 ElectroMAX DH10B E. coli ThermoFisher Cat#18290015 MCE membrane filter, 0.025 μm pore size Millipore Sigma Cat#VSWP02500 Chloramphenicol Millipore Sigma Cat#C0378 Electroporation cuvette (1 mm gap) VWR Cat#89047-206 SPRIselect beads Beckman Coulter Cat#B23317 GeneJET Plasmid Miniprep Kit ThermoFisher Cat#K0502 GeneJET Gel Extraction Kit ThermoFisher Cat#K0692 TransIT®-Lenti Transfection Reagent Millipore Sigma Cat#MIR6603 Quikchange Lightning Site-Directed Mutagenesis Kit Agilent Cat#210518 Genscript Genbuilder Gibson Mix Genscript Cat#L00701-50 Ligation sequencing DNA V14 Oxford Nanopore Technologies Cat#SQK-LSK114 Chromium GEM-X Single Cell 5 ′ Kit v3 10X Genomics Cat#PN-1000695 Chromium Next GEM Single Cell 5 ′ Kit v2 10X Genomics Cat#PN-1000263 (Continued on next page) e1 Immunity 59, 1–17.e1–e9, February 10, 2026 .. HEK293T cells (ATCC CRL-11268) were cultured in DMEM (ATCC) supplemented with 10% fetal bovine serum (FBS; Atlanta biologics) and penicillin-streptomycin (Gibco).
Ligation:Article Title: Scalable TCR synthesis and screening enable antigen reactivity mapping in vitiligo.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies APC anti-human CD69 (clone FN50) Biolegend Cat#310910; RRID: AB_314845 PE anti-human CD19 (clone HIB19) Biolegend Cat#302208; RRID: AB_314238 APC anti-human TCR α/β (clone IP26) Biolegend Cat#306718; RRID: AB_10612569 PE anti-human TCR α/β (clone IP26) Biolegend Cat#306708; RRID: AB_314646 Bacterial and virus strains Stbl3 chemical competent cells ThermoFisher Cat#C737303 ElectroMAX® DH10β electrocompetent E. coli ThermoFisher Cat#18290015 Biological samples Suction blister biopsies Vitiligo Clinic and Research Center, University of Massachusetts Chan Medical School N/A Chemicals, peptides, and recombinant proteins Synthesized peptides Genscript Table S2 Phorbol 12-myristate 13-acetate (PMA) ThermoFisher Cat#J63916.MA DEAE-dextran hydrochloride Sigma Cat#D9885-10G Ionomycin, 96% ThermoFisher Cat#J62448.MCR Critical commercial assays NEBNext Ultra II Q5 Master Mix NEB Cat#M0544L KAPA HiFi HotStart Readymix Roche Cat# KK2601 NEBridge Ligase Master Mix NEB Cat#M1100 NEBridge Golden Gate Assembly Kit (BsmBI-v2) NEB CatE1602 NEBridge Golden Gate Assembly Kit (BsaI-HF-v2) NEB Cat#E1601 BsaI-HF-v2 NEB Cat#R3733 BsmBI-v2 NEB Cat#R0739 BbsI-HF NEB Cat#R3539 SapI NEB Cat#R0569 FastDigest XbaI ThermoFisher Cat#FD0685 FastDigest Buffer ThermoFisher Cat#B64 Nuclease-free water (not DEPC-treated) ThermoFisher Cat#AM9937 Shrimp alkaline phosphatase (rSAP) NEB Cat#M0371 ElectroMAX DH10B E. coli ThermoFisher Cat#18290015 MCE membrane filter, 0.025 μm pore size Millipore Sigma Cat#VSWP02500 Chloramphenicol Millipore Sigma Cat#C0378 Electroporation cuvette (1 mm gap) VWR Cat#89047-206 SPRIselect beads Beckman Coulter Cat#B23317 GeneJET Plasmid Miniprep Kit ThermoFisher Cat#K0502 GeneJET Gel Extraction Kit ThermoFisher Cat#K0692 TransIT®-Lenti Transfection Reagent Millipore Sigma Cat#MIR6603 Quikchange Lightning Site-Directed Mutagenesis Kit Agilent Cat#210518 Genscript Genbuilder Gibson Mix Genscript Cat#L00701-50 Ligation sequencing DNA V14 Oxford Nanopore Technologies Cat#SQK-LSK114 Chromium GEM-X Single Cell 5 ′ Kit v3 10X Genomics Cat#PN-1000695 Chromium Next GEM Single Cell 5 ′ Kit v2 10X Genomics Cat#PN-1000263 (Continued on next page) e1 Immunity 59, 1–17.e1–e9, February 10, 2026 .. HEK293T cells (ATCC CRL-11268) were cultured in DMEM (ATCC) supplemented with 10% fetal bovine serum (FBS; Atlanta biologics) and penicillin-streptomycin (Gibco).
Sequencing:Article Title: Scalable TCR synthesis and screening enable antigen reactivity mapping in vitiligo.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies APC anti-human CD69 (clone FN50) Biolegend Cat#310910; RRID: AB_314845 PE anti-human CD19 (clone HIB19) Biolegend Cat#302208; RRID: AB_314238 APC anti-human TCR α/β (clone IP26) Biolegend Cat#306718; RRID: AB_10612569 PE anti-human TCR α/β (clone IP26) Biolegend Cat#306708; RRID: AB_314646 Bacterial and virus strains Stbl3 chemical competent cells ThermoFisher Cat#C737303 ElectroMAX® DH10β electrocompetent E. coli ThermoFisher Cat#18290015 Biological samples Suction blister biopsies Vitiligo Clinic and Research Center, University of Massachusetts Chan Medical School N/A Chemicals, peptides, and recombinant proteins Synthesized peptides Genscript Table S2 Phorbol 12-myristate 13-acetate (PMA) ThermoFisher Cat#J63916.MA DEAE-dextran hydrochloride Sigma Cat#D9885-10G Ionomycin, 96% ThermoFisher Cat#J62448.MCR Critical commercial assays NEBNext Ultra II Q5 Master Mix NEB Cat#M0544L KAPA HiFi HotStart Readymix Roche Cat# KK2601 NEBridge Ligase Master Mix NEB Cat#M1100 NEBridge Golden Gate Assembly Kit (BsmBI-v2) NEB CatE1602 NEBridge Golden Gate Assembly Kit (BsaI-HF-v2) NEB Cat#E1601 BsaI-HF-v2 NEB Cat#R3733 BsmBI-v2 NEB Cat#R0739 BbsI-HF NEB Cat#R3539 SapI NEB Cat#R0569 FastDigest XbaI ThermoFisher Cat#FD0685 FastDigest Buffer ThermoFisher Cat#B64 Nuclease-free water (not DEPC-treated) ThermoFisher Cat#AM9937 Shrimp alkaline phosphatase (rSAP) NEB Cat#M0371 ElectroMAX DH10B E. coli ThermoFisher Cat#18290015 MCE membrane filter, 0.025 μm pore size Millipore Sigma Cat#VSWP02500 Chloramphenicol Millipore Sigma Cat#C0378 Electroporation cuvette (1 mm gap) VWR Cat#89047-206 SPRIselect beads Beckman Coulter Cat#B23317 GeneJET Plasmid Miniprep Kit ThermoFisher Cat#K0502 GeneJET Gel Extraction Kit ThermoFisher Cat#K0692 TransIT®-Lenti Transfection Reagent Millipore Sigma Cat#MIR6603 Quikchange Lightning Site-Directed Mutagenesis Kit Agilent Cat#210518 Genscript Genbuilder Gibson Mix Genscript Cat#L00701-50 Ligation sequencing DNA V14 Oxford Nanopore Technologies Cat#SQK-LSK114 Chromium GEM-X Single Cell 5 ′ Kit v3 10X Genomics Cat#PN-1000695 Chromium Next GEM Single Cell 5 ′ Kit v2 10X Genomics Cat#PN-1000263 (Continued on next page) e1 Immunity 59, 1–17.e1–e9, February 10, 2026 .. HEK293T cells (ATCC CRL-11268) were cultured in DMEM (ATCC) supplemented with 10% fetal bovine serum (FBS; Atlanta biologics) and penicillin-streptomycin (Gibco).
Single Cell:Article Title: Scalable TCR synthesis and screening enable antigen reactivity mapping in vitiligo.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies APC anti-human CD69 (clone FN50) Biolegend Cat#310910; RRID: AB_314845 PE anti-human CD19 (clone HIB19) Biolegend Cat#302208; RRID: AB_314238 APC anti-human TCR α/β (clone IP26) Biolegend Cat#306718; RRID: AB_10612569 PE anti-human TCR α/β (clone IP26) Biolegend Cat#306708; RRID: AB_314646 Bacterial and virus strains Stbl3 chemical competent cells ThermoFisher Cat#C737303 ElectroMAX® DH10β electrocompetent E. coli ThermoFisher Cat#18290015 Biological samples Suction blister biopsies Vitiligo Clinic and Research Center, University of Massachusetts Chan Medical School N/A Chemicals, peptides, and recombinant proteins Synthesized peptides Genscript Table S2 Phorbol 12-myristate 13-acetate (PMA) ThermoFisher Cat#J63916.MA DEAE-dextran hydrochloride Sigma Cat#D9885-10G Ionomycin, 96% ThermoFisher Cat#J62448.MCR Critical commercial assays NEBNext Ultra II Q5 Master Mix NEB Cat#M0544L KAPA HiFi HotStart Readymix Roche Cat# KK2601 NEBridge Ligase Master Mix NEB Cat#M1100 NEBridge Golden Gate Assembly Kit (BsmBI-v2) NEB CatE1602 NEBridge Golden Gate Assembly Kit (BsaI-HF-v2) NEB Cat#E1601 BsaI-HF-v2 NEB Cat#R3733 BsmBI-v2 NEB Cat#R0739 BbsI-HF NEB Cat#R3539 SapI NEB Cat#R0569 FastDigest XbaI ThermoFisher Cat#FD0685 FastDigest Buffer ThermoFisher Cat#B64 Nuclease-free water (not DEPC-treated) ThermoFisher Cat#AM9937 Shrimp alkaline phosphatase (rSAP) NEB Cat#M0371 ElectroMAX DH10B E. coli ThermoFisher Cat#18290015 MCE membrane filter, 0.025 μm pore size Millipore Sigma Cat#VSWP02500 Chloramphenicol Millipore Sigma Cat#C0378 Electroporation cuvette (1 mm gap) VWR Cat#89047-206 SPRIselect beads Beckman Coulter Cat#B23317 GeneJET Plasmid Miniprep Kit ThermoFisher Cat#K0502 GeneJET Gel Extraction Kit ThermoFisher Cat#K0692 TransIT®-Lenti Transfection Reagent Millipore Sigma Cat#MIR6603 Quikchange Lightning Site-Directed Mutagenesis Kit Agilent Cat#210518 Genscript Genbuilder Gibson Mix Genscript Cat#L00701-50 Ligation sequencing DNA V14 Oxford Nanopore Technologies Cat#SQK-LSK114 Chromium GEM-X Single Cell 5 ′ Kit v3 10X Genomics Cat#PN-1000695 Chromium Next GEM Single Cell 5 ′ Kit v2 10X Genomics Cat#PN-1000263 (Continued on next page) e1 Immunity 59, 1–17.e1–e9, February 10, 2026 .. HEK293T cells (ATCC CRL-11268) were cultured in DMEM (ATCC) supplemented with 10% fetal bovine serum (FBS; Atlanta biologics) and penicillin-streptomycin (Gibco).
Purification:Article Title: An AI-driven workflow for the accelerated optimization of cell-free protein synthesis
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-6xHistidin-tag Sigma Cat#SAB2702220; RRID: AB_3095553 anti-GFP-tag Cell signaling Cat#2555; RRID: AB_10692764 anti-HiBiT-tag Promega Cat#N720A Bacterial and virus strains Escherichia coli DH5α Lab stock Escherichia coli BL21 (DE3) Lab stock Escherichia coli W3110 Lab stock Biological samples E. coli cell-free lysates Synthelis Biotech HeLa Lysate ThermoScientific Cat#88882 Chemicals, peptides, and recombinant proteins HEPES Sigma-Aldrich Cat#H3375 Nucleotide mix - Adenosine 5-triphosphate dipotassium salt hydrate Sigma-Aldrich Cat#A8937 Nucleotide mix - Guanosine 5ʹ-Triphosphate, Disodium Salt Sigma-Aldrich Cat#371701 Nucleotide mix - Sel disodique cytidine-5 ′ -triphosphate, 98+ % Thermo Scientific Chemicals Cat#J62238.ME Nucleotide mix - Uridine 5 ′ -triphosphate, sel trisodique, hydraté , 90% Thermo Scientific Chemicals Cat#226310010 3- PGA (Acide D-(− )-3-phosphoglycé rique disodium salt) Sigma-Aldrich Cat#P8877 Spermidine Sigma-Aldrich Cat#85558 Creatine phosphate crystalline, sup à 97% Sigma-Aldrich Cat#10621714001 PEG 8000 Sigma-Aldrich Cat#89510 Mg-glutamate Sigma-Aldrich Cat#49605 K-glutamate Alfa Aesar Cat#A17232 Amino acids - L-Alanine Sigma Cat#A7627 Amino acids - L-Arginine Sigma Cat#A5006 Amino acids - Glycine Sigma Cat#G7126 Amino acids - L-Histidine Merck Cat#1.04351.0100 Amino acids - L-Lysine Sigma Cat#L5626 Amino acids - L-Proline Sigma Cat#P0380 Amino acids - L-Serine Sigma Cat#S4500 Amino acids - L-Threonine Sigma Cat#T8625 Amino acids - L-Valine Sigma Cat#V0500 Amino acids - L-Aspartic Acid Sigma Cat#A9256 Amino acids - L-Asparagine anhydrous Sigma Cat#A0884 Amino acids - L-Cystine Sigma Cat#C8755 Amino acids - L-Glutamine Sigma Cat#G3126 Amino acids - L-Leucine Sigma Cat#L8000 Amino acids - L-Methionine Sigma Cat#64319 Amino acids - L-Tryptophan Sigma Cat#T8941 (Continued on next page) iScience 28, 113599, October 17, 2025 e1 .. REAGENT or RESOURCE SOURCE IDENTIFIER Amino acids - L-Tyrosine Sigma Cat#T3754 Amino acids - L-Phenylalanine Sigma Cat#78019 Amino acids - lsoleucine Sigma Cat#I7403 BsmBI-v2 NEB CatR0739S BbsI NEB Cat#R0539S BsaI NEB Cat#R3733S DpnI NEB Cat#R0176L BamHI Promega Cat#R6021 EcoRV Promega Cat#R6351 T4 DNA ligase Promega Cat#M1801 T4 DNA ligase NEB Cat#M0202 Ampicillin Sigma–Aldrich Cat#A1593 (IPTG) isopropyl -d-1-thiogalactopyranoside Sigma–Aldrich Cat#I5502 Critical commercial assays Q5 High-Fidelity 2X Master Mix NEB Cat#M0492L PCR purification kit NEB Cat#T1130 NucleoBond Xtra Maxi kit Macherey-Nagel Cat#740414.1 Nano-Glo® HiBiT Lytic Detection System Promega Cat#N3030 Nano-Glo® HiBiT Lytic Detection System Promega Cat#N3040 Deposited data icfree-ml source code https://github.com/brsynth/icfree-ml https://doi.org/10.5281/zenodo.14900104 icfree-ml conda packages https://anaconda.org/bioconda/icfree-ml https://doi.org/10.5281/zenodo.14893904 icfree-ml Galaxy wrappers https://toolshed.g2.bx.psu.edu https://doi.org/10.5281/zenodo.14893963 AI-CellFree - Init Galaxy workflow https://galaxy-synbiocad.org/ u/joan/w/icfree-init https://doi.org/10.5281/zenodo.14901023 AI-CellFree - Core Galaxy workflow https://galaxy-synbiocad.org/ u/joan/w/icfree-core https://doi.org/10.5281/zenodo.14899706 Experimental models: Organisms/strains Escherichia coli DH5α Escherichia coli BL21 (DE3) Escherichia coli W3110 Oligonucleotides See Table S3 Recombinant DNA Colicin M Synthesized by GeneCust Colicin E1 Synthesized by GeneCust GFP1-10 Synthesized by GeneCust GFP11 Synthesized by GeneCust His-GFP1-10 This study His-GFP11-Colicin M This study His-GFP11-Colicin E1 This study His-HiBiT-Colicin M This study His-HiBiT-Colicin E1 This study His-HiBiT-sfGFP This study Software and algorithms Echo Cherry Pick software Beckman Coulter Protocol Designer Opentrons Image Lab TM Software Bio-Rad Laboratories ChatGPT-4 Openai e2 iScience 28, 113599, October 17, 2025 ..
Software:Article Title: An AI-driven workflow for the accelerated optimization of cell-free protein synthesis
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-6xHistidin-tag Sigma Cat#SAB2702220; RRID: AB_3095553 anti-GFP-tag Cell signaling Cat#2555; RRID: AB_10692764 anti-HiBiT-tag Promega Cat#N720A Bacterial and virus strains Escherichia coli DH5α Lab stock Escherichia coli BL21 (DE3) Lab stock Escherichia coli W3110 Lab stock Biological samples E. coli cell-free lysates Synthelis Biotech HeLa Lysate ThermoScientific Cat#88882 Chemicals, peptides, and recombinant proteins HEPES Sigma-Aldrich Cat#H3375 Nucleotide mix - Adenosine 5-triphosphate dipotassium salt hydrate Sigma-Aldrich Cat#A8937 Nucleotide mix - Guanosine 5ʹ-Triphosphate, Disodium Salt Sigma-Aldrich Cat#371701 Nucleotide mix - Sel disodique cytidine-5 ′ -triphosphate, 98+ % Thermo Scientific Chemicals Cat#J62238.ME Nucleotide mix - Uridine 5 ′ -triphosphate, sel trisodique, hydraté , 90% Thermo Scientific Chemicals Cat#226310010 3- PGA (Acide D-(− )-3-phosphoglycé rique disodium salt) Sigma-Aldrich Cat#P8877 Spermidine Sigma-Aldrich Cat#85558 Creatine phosphate crystalline, sup à 97% Sigma-Aldrich Cat#10621714001 PEG 8000 Sigma-Aldrich Cat#89510 Mg-glutamate Sigma-Aldrich Cat#49605 K-glutamate Alfa Aesar Cat#A17232 Amino acids - L-Alanine Sigma Cat#A7627 Amino acids - L-Arginine Sigma Cat#A5006 Amino acids - Glycine Sigma Cat#G7126 Amino acids - L-Histidine Merck Cat#1.04351.0100 Amino acids - L-Lysine Sigma Cat#L5626 Amino acids - L-Proline Sigma Cat#P0380 Amino acids - L-Serine Sigma Cat#S4500 Amino acids - L-Threonine Sigma Cat#T8625 Amino acids - L-Valine Sigma Cat#V0500 Amino acids - L-Aspartic Acid Sigma Cat#A9256 Amino acids - L-Asparagine anhydrous Sigma Cat#A0884 Amino acids - L-Cystine Sigma Cat#C8755 Amino acids - L-Glutamine Sigma Cat#G3126 Amino acids - L-Leucine Sigma Cat#L8000 Amino acids - L-Methionine Sigma Cat#64319 Amino acids - L-Tryptophan Sigma Cat#T8941 (Continued on next page) iScience 28, 113599, October 17, 2025 e1 .. REAGENT or RESOURCE SOURCE IDENTIFIER Amino acids - L-Tyrosine Sigma Cat#T3754 Amino acids - L-Phenylalanine Sigma Cat#78019 Amino acids - lsoleucine Sigma Cat#I7403 BsmBI-v2 NEB CatR0739S BbsI NEB Cat#R0539S BsaI NEB Cat#R3733S DpnI NEB Cat#R0176L BamHI Promega Cat#R6021 EcoRV Promega Cat#R6351 T4 DNA ligase Promega Cat#M1801 T4 DNA ligase NEB Cat#M0202 Ampicillin Sigma–Aldrich Cat#A1593 (IPTG) isopropyl -d-1-thiogalactopyranoside Sigma–Aldrich Cat#I5502 Critical commercial assays Q5 High-Fidelity 2X Master Mix NEB Cat#M0492L PCR purification kit NEB Cat#T1130 NucleoBond Xtra Maxi kit Macherey-Nagel Cat#740414.1 Nano-Glo® HiBiT Lytic Detection System Promega Cat#N3030 Nano-Glo® HiBiT Lytic Detection System Promega Cat#N3040 Deposited data icfree-ml source code https://github.com/brsynth/icfree-ml https://doi.org/10.5281/zenodo.14900104 icfree-ml conda packages https://anaconda.org/bioconda/icfree-ml https://doi.org/10.5281/zenodo.14893904 icfree-ml Galaxy wrappers https://toolshed.g2.bx.psu.edu https://doi.org/10.5281/zenodo.14893963 AI-CellFree - Init Galaxy workflow https://galaxy-synbiocad.org/ u/joan/w/icfree-init https://doi.org/10.5281/zenodo.14901023 AI-CellFree - Core Galaxy workflow https://galaxy-synbiocad.org/ u/joan/w/icfree-core https://doi.org/10.5281/zenodo.14899706 Experimental models: Organisms/strains Escherichia coli DH5α Escherichia coli BL21 (DE3) Escherichia coli W3110 Oligonucleotides See Table S3 Recombinant DNA Colicin M Synthesized by GeneCust Colicin E1 Synthesized by GeneCust GFP1-10 Synthesized by GeneCust GFP11 Synthesized by GeneCust His-GFP1-10 This study His-GFP11-Colicin M This study His-GFP11-Colicin E1 This study His-HiBiT-Colicin M This study His-HiBiT-Colicin E1 This study His-HiBiT-sfGFP This study Software and algorithms Echo Cherry Pick software Beckman Coulter Protocol Designer Opentrons Image Lab TM Software Bio-Rad Laboratories ChatGPT-4 Openai e2 iScience 28, 113599, October 17, 2025 ..
Article Title: Protocol to implement saturation mutagenesis-reinforced functional assays to resolve small-sized variants in disease-related genes.
Article Snippet: .. Continued REAGENT or RESOURCE SOURCE IDENTIFIER Viobility 405/452 fixable dye Miltenyi Biotec Cat#130-130-420 MACS BSA stock solution Miltenyi Biotec Cat#130-091-376 autoMACS rinsing solution Miltenyi Biotec Cat#130-091-222 Human BD Fc Block BD Pharmingen Cat#564220 Q5 high-fidelity DNA polymerase NEB Cat#M0491SVIAL Q5 reaction buffer NEB Cat#B9027SVIAL Q5 High GC Enhancer NEB Cat#B9028AVIAL dNTPs NEB Cat#N0447 BsmBI-v2 NEB CatR0739S BsaI-HFv2 NEB Cat#R3733S XbaI NEB Cat#R0145S BamHI-HF NEB Cat#R3136S Type2M enzyme MspJI NEB Cat#R0661S Type2M enzyme DpnI NEB Cat#R0176S Polybrene Sigma-Aldrich Cat#107689 Critical commercial assays Synthetic single guide RNA kit Synthego N/A NucleoSpin gel and PCR clean-up kit Takara Cat#740609 PureLink midiprep kit Invitrogen Cat#K210014 PureLink genomic DNA mini kit Invitrogen Cat#K182002 SE Cell Line 4D-Nucleofector X Kit S Lonza Cat#V4XC-1032 Experimental models: Cell lines HAP1 Horizon Discovery C631 HEK293T ATCC CRL-3216 MB135 Dr. Stephen J. Tapscott N/A Recombinant DNA Lenti-UbC-FKRP-EF1a-BSD Ma et al. 1 Addgene Plasmid #205150 psPAX2 Dr. Didier Trono Addgene Plasmid #12260 pMD2.G Dr. Didier Trono Addgene Plasmid #12259 Software and algorithms R R Core Team https://www.r-project.org/contributors.html Scripts for oligo design, pre-SMuRF experiments Ma et al. 1 https://doi.org/10.5281/zenodo.13328292 Raw NGS data analytical pipeline Ma et al. 1 https://doi.org/10.5281/zenodo.13328302 Scripts for generating SMuRF scores and downstream analyses Ma et al. 1 https://doi.org/10.5281/zenodo.13328286 Other 0.45 μm PES filter Thermo Scientific Cat#165-0045 40 μm cell strainer Falcon Cat#352340 3D Rocker Benchmark Scientific Cat#B3D1008 Nikon microscope object marker Fisher Scientific Cat#NIKON-MBW10020 10 3 10 mm PYREX cloning cylinders Avantor/VWR Cat#22877-256 TC20 automated cell counter Bio-Rad Cat#1450102 Qubit fluorometer Thermo Scientific Cat#Q33226 STAR Protocols 6, 103909, September 19, 2025 3 Note: Synthego Synthetic Single Guide RNA (sgRNA) Kits can be utilized for sgRNA design and synthesis. ..
Blocking Assay:Article Title: Protocol to implement saturation mutagenesis-reinforced functional assays to resolve small-sized variants in disease-related genes.
Article Snippet: .. Continued REAGENT or RESOURCE SOURCE IDENTIFIER Viobility 405/452 fixable dye Miltenyi Biotec Cat#130-130-420 MACS BSA stock solution Miltenyi Biotec Cat#130-091-376 autoMACS rinsing solution Miltenyi Biotec Cat#130-091-222 Human BD Fc Block BD Pharmingen Cat#564220 Q5 high-fidelity DNA polymerase NEB Cat#M0491SVIAL Q5 reaction buffer NEB Cat#B9027SVIAL Q5 High GC Enhancer NEB Cat#B9028AVIAL dNTPs NEB Cat#N0447 BsmBI-v2 NEB CatR0739S BsaI-HFv2 NEB Cat#R3733S XbaI NEB Cat#R0145S BamHI-HF NEB Cat#R3136S Type2M enzyme MspJI NEB Cat#R0661S Type2M enzyme DpnI NEB Cat#R0176S Polybrene Sigma-Aldrich Cat#107689 Critical commercial assays Synthetic single guide RNA kit Synthego N/A NucleoSpin gel and PCR clean-up kit Takara Cat#740609 PureLink midiprep kit Invitrogen Cat#K210014 PureLink genomic DNA mini kit Invitrogen Cat#K182002 SE Cell Line 4D-Nucleofector X Kit S Lonza Cat#V4XC-1032 Experimental models: Cell lines HAP1 Horizon Discovery C631 HEK293T ATCC CRL-3216 MB135 Dr. Stephen J. Tapscott N/A Recombinant DNA Lenti-UbC-FKRP-EF1a-BSD Ma et al. 1 Addgene Plasmid #205150 psPAX2 Dr. Didier Trono Addgene Plasmid #12260 pMD2.G Dr. Didier Trono Addgene Plasmid #12259 Software and algorithms R R Core Team https://www.r-project.org/contributors.html Scripts for oligo design, pre-SMuRF experiments Ma et al. 1 https://doi.org/10.5281/zenodo.13328292 Raw NGS data analytical pipeline Ma et al. 1 https://doi.org/10.5281/zenodo.13328302 Scripts for generating SMuRF scores and downstream analyses Ma et al. 1 https://doi.org/10.5281/zenodo.13328286 Other 0.45 μm PES filter Thermo Scientific Cat#165-0045 40 μm cell strainer Falcon Cat#352340 3D Rocker Benchmark Scientific Cat#B3D1008 Nikon microscope object marker Fisher Scientific Cat#NIKON-MBW10020 10 3 10 mm PYREX cloning cylinders Avantor/VWR Cat#22877-256 TC20 automated cell counter Bio-Rad Cat#1450102 Qubit fluorometer Thermo Scientific Cat#Q33226 STAR Protocols 6, 103909, September 19, 2025 3 Note: Synthego Synthetic Single Guide RNA (sgRNA) Kits can be utilized for sgRNA design and synthesis. ..
Next-Generation Sequencing:Article Title: Protocol to implement saturation mutagenesis-reinforced functional assays to resolve small-sized variants in disease-related genes.
Article Snippet: .. Continued REAGENT or RESOURCE SOURCE IDENTIFIER Viobility 405/452 fixable dye Miltenyi Biotec Cat#130-130-420 MACS BSA stock solution Miltenyi Biotec Cat#130-091-376 autoMACS rinsing solution Miltenyi Biotec Cat#130-091-222 Human BD Fc Block BD Pharmingen Cat#564220 Q5 high-fidelity DNA polymerase NEB Cat#M0491SVIAL Q5 reaction buffer NEB Cat#B9027SVIAL Q5 High GC Enhancer NEB Cat#B9028AVIAL dNTPs NEB Cat#N0447 BsmBI-v2 NEB CatR0739S BsaI-HFv2 NEB Cat#R3733S XbaI NEB Cat#R0145S BamHI-HF NEB Cat#R3136S Type2M enzyme MspJI NEB Cat#R0661S Type2M enzyme DpnI NEB Cat#R0176S Polybrene Sigma-Aldrich Cat#107689 Critical commercial assays Synthetic single guide RNA kit Synthego N/A NucleoSpin gel and PCR clean-up kit Takara Cat#740609 PureLink midiprep kit Invitrogen Cat#K210014 PureLink genomic DNA mini kit Invitrogen Cat#K182002 SE Cell Line 4D-Nucleofector X Kit S Lonza Cat#V4XC-1032 Experimental models: Cell lines HAP1 Horizon Discovery C631 HEK293T ATCC CRL-3216 MB135 Dr. Stephen J. Tapscott N/A Recombinant DNA Lenti-UbC-FKRP-EF1a-BSD Ma et al. 1 Addgene Plasmid #205150 psPAX2 Dr. Didier Trono Addgene Plasmid #12260 pMD2.G Dr. Didier Trono Addgene Plasmid #12259 Software and algorithms R R Core Team https://www.r-project.org/contributors.html Scripts for oligo design, pre-SMuRF experiments Ma et al. 1 https://doi.org/10.5281/zenodo.13328292 Raw NGS data analytical pipeline Ma et al. 1 https://doi.org/10.5281/zenodo.13328302 Scripts for generating SMuRF scores and downstream analyses Ma et al. 1 https://doi.org/10.5281/zenodo.13328286 Other 0.45 μm PES filter Thermo Scientific Cat#165-0045 40 μm cell strainer Falcon Cat#352340 3D Rocker Benchmark Scientific Cat#B3D1008 Nikon microscope object marker Fisher Scientific Cat#NIKON-MBW10020 10 3 10 mm PYREX cloning cylinders Avantor/VWR Cat#22877-256 TC20 automated cell counter Bio-Rad Cat#1450102 Qubit fluorometer Thermo Scientific Cat#Q33226 STAR Protocols 6, 103909, September 19, 2025 3 Note: Synthego Synthetic Single Guide RNA (sgRNA) Kits can be utilized for sgRNA design and synthesis. ..
Microscopy:Article Title: Protocol to implement saturation mutagenesis-reinforced functional assays to resolve small-sized variants in disease-related genes.
Article Snippet: .. Continued REAGENT or RESOURCE SOURCE IDENTIFIER Viobility 405/452 fixable dye Miltenyi Biotec Cat#130-130-420 MACS BSA stock solution Miltenyi Biotec Cat#130-091-376 autoMACS rinsing solution Miltenyi Biotec Cat#130-091-222 Human BD Fc Block BD Pharmingen Cat#564220 Q5 high-fidelity DNA polymerase NEB Cat#M0491SVIAL Q5 reaction buffer NEB Cat#B9027SVIAL Q5 High GC Enhancer NEB Cat#B9028AVIAL dNTPs NEB Cat#N0447 BsmBI-v2 NEB CatR0739S BsaI-HFv2 NEB Cat#R3733S XbaI NEB Cat#R0145S BamHI-HF NEB Cat#R3136S Type2M enzyme MspJI NEB Cat#R0661S Type2M enzyme DpnI NEB Cat#R0176S Polybrene Sigma-Aldrich Cat#107689 Critical commercial assays Synthetic single guide RNA kit Synthego N/A NucleoSpin gel and PCR clean-up kit Takara Cat#740609 PureLink midiprep kit Invitrogen Cat#K210014 PureLink genomic DNA mini kit Invitrogen Cat#K182002 SE Cell Line 4D-Nucleofector X Kit S Lonza Cat#V4XC-1032 Experimental models: Cell lines HAP1 Horizon Discovery C631 HEK293T ATCC CRL-3216 MB135 Dr. Stephen J. Tapscott N/A Recombinant DNA Lenti-UbC-FKRP-EF1a-BSD Ma et al. 1 Addgene Plasmid #205150 psPAX2 Dr. Didier Trono Addgene Plasmid #12260 pMD2.G Dr. Didier Trono Addgene Plasmid #12259 Software and algorithms R R Core Team https://www.r-project.org/contributors.html Scripts for oligo design, pre-SMuRF experiments Ma et al. 1 https://doi.org/10.5281/zenodo.13328292 Raw NGS data analytical pipeline Ma et al. 1 https://doi.org/10.5281/zenodo.13328302 Scripts for generating SMuRF scores and downstream analyses Ma et al. 1 https://doi.org/10.5281/zenodo.13328286 Other 0.45 μm PES filter Thermo Scientific Cat#165-0045 40 μm cell strainer Falcon Cat#352340 3D Rocker Benchmark Scientific Cat#B3D1008 Nikon microscope object marker Fisher Scientific Cat#NIKON-MBW10020 10 3 10 mm PYREX cloning cylinders Avantor/VWR Cat#22877-256 TC20 automated cell counter Bio-Rad Cat#1450102 Qubit fluorometer Thermo Scientific Cat#Q33226 STAR Protocols 6, 103909, September 19, 2025 3 Note: Synthego Synthetic Single Guide RNA (sgRNA) Kits can be utilized for sgRNA design and synthesis. ..
Marker:Article Title: Protocol to implement saturation mutagenesis-reinforced functional assays to resolve small-sized variants in disease-related genes.
Article Snippet: .. Continued REAGENT or RESOURCE SOURCE IDENTIFIER Viobility 405/452 fixable dye Miltenyi Biotec Cat#130-130-420 MACS BSA stock solution Miltenyi Biotec Cat#130-091-376 autoMACS rinsing solution Miltenyi Biotec Cat#130-091-222 Human BD Fc Block BD Pharmingen Cat#564220 Q5 high-fidelity DNA polymerase NEB Cat#M0491SVIAL Q5 reaction buffer NEB Cat#B9027SVIAL Q5 High GC Enhancer NEB Cat#B9028AVIAL dNTPs NEB Cat#N0447 BsmBI-v2 NEB CatR0739S BsaI-HFv2 NEB Cat#R3733S XbaI NEB Cat#R0145S BamHI-HF NEB Cat#R3136S Type2M enzyme MspJI NEB Cat#R0661S Type2M enzyme DpnI NEB Cat#R0176S Polybrene Sigma-Aldrich Cat#107689 Critical commercial assays Synthetic single guide RNA kit Synthego N/A NucleoSpin gel and PCR clean-up kit Takara Cat#740609 PureLink midiprep kit Invitrogen Cat#K210014 PureLink genomic DNA mini kit Invitrogen Cat#K182002 SE Cell Line 4D-Nucleofector X Kit S Lonza Cat#V4XC-1032 Experimental models: Cell lines HAP1 Horizon Discovery C631 HEK293T ATCC CRL-3216 MB135 Dr. Stephen J. Tapscott N/A Recombinant DNA Lenti-UbC-FKRP-EF1a-BSD Ma et al. 1 Addgene Plasmid #205150 psPAX2 Dr. Didier Trono Addgene Plasmid #12260 pMD2.G Dr. Didier Trono Addgene Plasmid #12259 Software and algorithms R R Core Team https://www.r-project.org/contributors.html Scripts for oligo design, pre-SMuRF experiments Ma et al. 1 https://doi.org/10.5281/zenodo.13328292 Raw NGS data analytical pipeline Ma et al. 1 https://doi.org/10.5281/zenodo.13328302 Scripts for generating SMuRF scores and downstream analyses Ma et al. 1 https://doi.org/10.5281/zenodo.13328286 Other 0.45 μm PES filter Thermo Scientific Cat#165-0045 40 μm cell strainer Falcon Cat#352340 3D Rocker Benchmark Scientific Cat#B3D1008 Nikon microscope object marker Fisher Scientific Cat#NIKON-MBW10020 10 3 10 mm PYREX cloning cylinders Avantor/VWR Cat#22877-256 TC20 automated cell counter Bio-Rad Cat#1450102 Qubit fluorometer Thermo Scientific Cat#Q33226 STAR Protocols 6, 103909, September 19, 2025 3 Note: Synthego Synthetic Single Guide RNA (sgRNA) Kits can be utilized for sgRNA design and synthesis. ..
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