Reverse Transcription:Article Title: Homologous recombination mutants cause differing lethality between h- and h+ Schizosaccharomyces pombe strains due to mat1 heterochromatin.
Article Snippet: For reverse transcription, 0.5 lg RNA was mixed with 0.5 lL of 100 lM oligo(dT)20 primer, 0.5 lL of 100 lM random hexamers (Generi Biotech, Hradec Kr alov e, Czech Republic), 1 lL of 10 mM dNTP mix (NEB), and water to 8.3 lL total. .. The mixture was denatured for 5 min at 65 °C, cooled on ice, and supplemented with 0.5 lL M-MuLV Reverse Transcriptase (NEB), 1 lL 109 RT buffer, and 0.2 lL SUPERase-In RNase Inhibitor (Invitrogen, Thermo Fisher Scientific). ..
Article Title: PUS7 pseudouridylation of 7SK orchestrates human somatic cell reprogramming into iPS cells.
Article Snippet: .. For RT and splint ligation, 1 μl of 10× annealing buffer (250 mM Tris-HCl pH 7.4 and 480 mM KCl) and 1 μl of 0.5 μM RT primer (5′-CCA CAT GCA GCG CCT CAT TTG G-3′) were added and the mixture was heated to 93 °C for 2 min. Then, 5 μl of 2× AMV reverse transcriptase reaction mix was added and the RT reaction was performed at 42 °C for 1 h, followed by 85 °C for 5 min. To degrade RNA, 1 μl of RNase H (New England Biolabs, M0297L) was added and incubated at 37 °C for 20 min and then at 85 °C for 5 min. For splint ligation, 1 μl of 1.5 μM adaptor/splint oligo mix (adaptor, 5′-/5Phos/CCA TGG ACC GAA GAC CGG TCC TCC TCT A-3′; splint, 5′-CGG TCC ATG GAG GAG AAC GT/3SpC3/-3′) was added and heated to 75 °C for 3 min. .. Subsequently, T4 DNA ligase (New England Biolabs, M0202L) and 1× ligase buffer with 12.5% DMSO were added and ligation was performed at 16 °C for 16 h, followed by heat inactivation at 65 °C for 10 min. Lastly, 2 μl of the reaction mixture was used as a template for PCR amplification (35 cycles) using the forward primer 5′-TAG AGG AGG ACC GGT CTT CGG TC-3′ and the RT primer as the reverse primer.
Article Title: Cotranslational membrane protein biogenesis by an EMC-bound translocon.
Article Snippet: After adding Nature Structural & Molecular Biology Article https://doi.org/10.1038/s41594-026-01889-2 100 μl of chloroform (Sigma-Aldrich, cat. no. C2432), RNA in the aqueous layer was separated by centrifugation at 12,000g for 15 min at 4 °C and precipitated overnight by adding 0.1 volume of 3 M sodium acetate (Invitrogen, cat. no. AM9740), 20 μg of GlycoBlue (Invitrogen, cat. no. AM9515) and 1.2 volumes of isopropanol. .. Purified RNase footprints used for DNA library preparation were resuspended in 5 μl of T4 polynucleotide kinase (PNK) reaction mixture (1× PNK buffer, 10 U of PNK (NEB, cat. no. M0201L) and 10 U of SUPERaseIn (Invitrogen, cat. no. AM2694)) and end-repaired at 37 °C for 1 h. Next, 5 μl of polyadenylation mix containing 10 U of Escherichia coli poly (A) polymerase (NEB, cat. no. M0276L), 2 μl of 5× first-strand buffer (250 mM Tris-HCl pH 8.3, 375 mM KCl, 15 mM MgCl2), 0.25 mM ATP and 10 U SUPERaseIn was added, and the reaction was incubated at 37 °C for 2 h. The resulting polyadenylated RNAs were reverse transcribed by SMART-RT, by adding 2.5 μl of reverse transcription primer (10 μM, 5′-ACGTGTGCTCTTCCGATCTNNNNNN NNNNNNNNNTTTTTTTTTTTTTTTVN-3′, where ‘N’ is any base and ‘V’ is ‘A’, ‘C’ or ‘G’), annealing by heating to 72 °C for 5 min and cooling on ice, and then supplementing with 17.6 μl of SMART-RT mixture (2.5 μl of dNTP (10 mM), 400 U of Superscript II reverse transcriptase (Invitrogen, cat. no. 18064014), 10 U of SUPERaseIn, 4 μl of Superscript II first-strand buffer (5×), 1.5 μl of DTT (100 mM), 6 μl of Betaine (5 M), 0.12 μl of MgCl2 (1 M), 0.5 μl of template-switching oligos (100 μM 5′-CTCTTTCCCTACACGACGCTCTTCCGATCTNNNNrGrG+G-3′, where ‘N’ is any base and ‘+G’ is a locked nucleic acid guanylate) and 0.5 μl of nuclease-free water). ..
Recombinant:Article Title: Reconstructing the west-east genetic division in Indonesia using ancient genomes
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Biological samples Ancient skeletal element This paper LMCM1 Ancient skeletal element This paper LMCM2 Chemicals, peptides, and recombinant proteins T4 Polynucleotide Kinase (10 U/uL) ThermoFisher EK0032 KAPA HiFi HS Uracil +ready Mix KAPA Biosystems KK2802 ATP (100 mM) Fermentas R0441 dNTP Mix (25 mM each) ThermoFisher R1121 Dynabeads MyOne Streptavidin C1 InvitrogenTM 65001 AccuPrime Pfx DNA polymerase ThermoFisher 12344024 T4 RNA ligase reaction buffer(10x) NEB B0216L FastAP thermosensitive alkaline phosphatase(1U/μL) ThermoFisher EF0651 T4 DNA ligase, 5 U/μL ThermoFisher EL0012 T4 DNA ligase, high concentrated 30 U/μL ThermoFisher EL0013 Klenow fragment (10 U/uL) ThermoFisher EP0052 PB buffer Qiagen 19066 AMPure XP Beads Beckman A63882 Isopropyl alcohol Macklin I811932-4L Anhydrous ethanol, pharmaceutical grade, 99.5% Mreda M206333-5L 5M NaCl Sigma-AldriCE S5150-1L Nuclease-free water Thermo Fisher 4387936 20× SSC buffer Sigma-AldriCE S6639-1L 3 M Sodium acetate, pH 5.2 Sigma-AldriCE S7899 Ethanol (Analytical Reagent) Sigma-AldriCE 459844 20% (wt/vol) SDS solution Thermo Scientific J63394.AK Tris (1 M)、pH 8.0、no RNase Thermo Scientific AM9856 Guanidine hydrochloride Sigma-Aldrich G3272-2 kg Water, HPLC grade Sigma-Aldrich AH365-4HC Proteinase K (10 mg/mL) Sigma-Aldrich P6556 0.5M EDTA (pH8.0) Applichen A48921000 NaClO Sinopharm 80010428 Agarose Biowest 111860 Critical commercial assays Qubit dsDNA Quantitation Kit Invitrogen Q32854 MinElute PCR Purification Kit QIAGEN 28006 Twist Mitochondrial Panel Kit Twist 102040 Twist Binding and Purification Beads Twist 100984 Twist Universal Blockers Twist 100767 Twist Hybridization Reagents Twist 100982 Twist Wash Buffers Twist 100846 Twist Ancient Human DNA Panel Twist 106658 Deposited data BAM files reported in this paper have been deposited in the GSA-Human (https://ngdc. cncb.ac.cn/gsa-human/). ..
High Performance Liquid Chromatography:Article Title: Reconstructing the west-east genetic division in Indonesia using ancient genomes
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Biological samples Ancient skeletal element This paper LMCM1 Ancient skeletal element This paper LMCM2 Chemicals, peptides, and recombinant proteins T4 Polynucleotide Kinase (10 U/uL) ThermoFisher EK0032 KAPA HiFi HS Uracil +ready Mix KAPA Biosystems KK2802 ATP (100 mM) Fermentas R0441 dNTP Mix (25 mM each) ThermoFisher R1121 Dynabeads MyOne Streptavidin C1 InvitrogenTM 65001 AccuPrime Pfx DNA polymerase ThermoFisher 12344024 T4 RNA ligase reaction buffer(10x) NEB B0216L FastAP thermosensitive alkaline phosphatase(1U/μL) ThermoFisher EF0651 T4 DNA ligase, 5 U/μL ThermoFisher EL0012 T4 DNA ligase, high concentrated 30 U/μL ThermoFisher EL0013 Klenow fragment (10 U/uL) ThermoFisher EP0052 PB buffer Qiagen 19066 AMPure XP Beads Beckman A63882 Isopropyl alcohol Macklin I811932-4L Anhydrous ethanol, pharmaceutical grade, 99.5% Mreda M206333-5L 5M NaCl Sigma-AldriCE S5150-1L Nuclease-free water Thermo Fisher 4387936 20× SSC buffer Sigma-AldriCE S6639-1L 3 M Sodium acetate, pH 5.2 Sigma-AldriCE S7899 Ethanol (Analytical Reagent) Sigma-AldriCE 459844 20% (wt/vol) SDS solution Thermo Scientific J63394.AK Tris (1 M)、pH 8.0、no RNase Thermo Scientific AM9856 Guanidine hydrochloride Sigma-Aldrich G3272-2 kg Water, HPLC grade Sigma-Aldrich AH365-4HC Proteinase K (10 mg/mL) Sigma-Aldrich P6556 0.5M EDTA (pH8.0) Applichen A48921000 NaClO Sinopharm 80010428 Agarose Biowest 111860 Critical commercial assays Qubit dsDNA Quantitation Kit Invitrogen Q32854 MinElute PCR Purification Kit QIAGEN 28006 Twist Mitochondrial Panel Kit Twist 102040 Twist Binding and Purification Beads Twist 100984 Twist Universal Blockers Twist 100767 Twist Hybridization Reagents Twist 100982 Twist Wash Buffers Twist 100846 Twist Ancient Human DNA Panel Twist 106658 Deposited data BAM files reported in this paper have been deposited in the GSA-Human (https://ngdc. cncb.ac.cn/gsa-human/). ..
Quantitation Assay:Article Title: Reconstructing the west-east genetic division in Indonesia using ancient genomes
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Biological samples Ancient skeletal element This paper LMCM1 Ancient skeletal element This paper LMCM2 Chemicals, peptides, and recombinant proteins T4 Polynucleotide Kinase (10 U/uL) ThermoFisher EK0032 KAPA HiFi HS Uracil +ready Mix KAPA Biosystems KK2802 ATP (100 mM) Fermentas R0441 dNTP Mix (25 mM each) ThermoFisher R1121 Dynabeads MyOne Streptavidin C1 InvitrogenTM 65001 AccuPrime Pfx DNA polymerase ThermoFisher 12344024 T4 RNA ligase reaction buffer(10x) NEB B0216L FastAP thermosensitive alkaline phosphatase(1U/μL) ThermoFisher EF0651 T4 DNA ligase, 5 U/μL ThermoFisher EL0012 T4 DNA ligase, high concentrated 30 U/μL ThermoFisher EL0013 Klenow fragment (10 U/uL) ThermoFisher EP0052 PB buffer Qiagen 19066 AMPure XP Beads Beckman A63882 Isopropyl alcohol Macklin I811932-4L Anhydrous ethanol, pharmaceutical grade, 99.5% Mreda M206333-5L 5M NaCl Sigma-AldriCE S5150-1L Nuclease-free water Thermo Fisher 4387936 20× SSC buffer Sigma-AldriCE S6639-1L 3 M Sodium acetate, pH 5.2 Sigma-AldriCE S7899 Ethanol (Analytical Reagent) Sigma-AldriCE 459844 20% (wt/vol) SDS solution Thermo Scientific J63394.AK Tris (1 M)、pH 8.0、no RNase Thermo Scientific AM9856 Guanidine hydrochloride Sigma-Aldrich G3272-2 kg Water, HPLC grade Sigma-Aldrich AH365-4HC Proteinase K (10 mg/mL) Sigma-Aldrich P6556 0.5M EDTA (pH8.0) Applichen A48921000 NaClO Sinopharm 80010428 Agarose Biowest 111860 Critical commercial assays Qubit dsDNA Quantitation Kit Invitrogen Q32854 MinElute PCR Purification Kit QIAGEN 28006 Twist Mitochondrial Panel Kit Twist 102040 Twist Binding and Purification Beads Twist 100984 Twist Universal Blockers Twist 100767 Twist Hybridization Reagents Twist 100982 Twist Wash Buffers Twist 100846 Twist Ancient Human DNA Panel Twist 106658 Deposited data BAM files reported in this paper have been deposited in the GSA-Human (https://ngdc. cncb.ac.cn/gsa-human/). ..
Polymerase Chain Reaction:Article Title: Reconstructing the west-east genetic division in Indonesia using ancient genomes
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Biological samples Ancient skeletal element This paper LMCM1 Ancient skeletal element This paper LMCM2 Chemicals, peptides, and recombinant proteins T4 Polynucleotide Kinase (10 U/uL) ThermoFisher EK0032 KAPA HiFi HS Uracil +ready Mix KAPA Biosystems KK2802 ATP (100 mM) Fermentas R0441 dNTP Mix (25 mM each) ThermoFisher R1121 Dynabeads MyOne Streptavidin C1 InvitrogenTM 65001 AccuPrime Pfx DNA polymerase ThermoFisher 12344024 T4 RNA ligase reaction buffer(10x) NEB B0216L FastAP thermosensitive alkaline phosphatase(1U/μL) ThermoFisher EF0651 T4 DNA ligase, 5 U/μL ThermoFisher EL0012 T4 DNA ligase, high concentrated 30 U/μL ThermoFisher EL0013 Klenow fragment (10 U/uL) ThermoFisher EP0052 PB buffer Qiagen 19066 AMPure XP Beads Beckman A63882 Isopropyl alcohol Macklin I811932-4L Anhydrous ethanol, pharmaceutical grade, 99.5% Mreda M206333-5L 5M NaCl Sigma-AldriCE S5150-1L Nuclease-free water Thermo Fisher 4387936 20× SSC buffer Sigma-AldriCE S6639-1L 3 M Sodium acetate, pH 5.2 Sigma-AldriCE S7899 Ethanol (Analytical Reagent) Sigma-AldriCE 459844 20% (wt/vol) SDS solution Thermo Scientific J63394.AK Tris (1 M)、pH 8.0、no RNase Thermo Scientific AM9856 Guanidine hydrochloride Sigma-Aldrich G3272-2 kg Water, HPLC grade Sigma-Aldrich AH365-4HC Proteinase K (10 mg/mL) Sigma-Aldrich P6556 0.5M EDTA (pH8.0) Applichen A48921000 NaClO Sinopharm 80010428 Agarose Biowest 111860 Critical commercial assays Qubit dsDNA Quantitation Kit Invitrogen Q32854 MinElute PCR Purification Kit QIAGEN 28006 Twist Mitochondrial Panel Kit Twist 102040 Twist Binding and Purification Beads Twist 100984 Twist Universal Blockers Twist 100767 Twist Hybridization Reagents Twist 100982 Twist Wash Buffers Twist 100846 Twist Ancient Human DNA Panel Twist 106658 Deposited data BAM files reported in this paper have been deposited in the GSA-Human (https://ngdc. cncb.ac.cn/gsa-human/). ..
Purification:Article Title: Reconstructing the west-east genetic division in Indonesia using ancient genomes
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Biological samples Ancient skeletal element This paper LMCM1 Ancient skeletal element This paper LMCM2 Chemicals, peptides, and recombinant proteins T4 Polynucleotide Kinase (10 U/uL) ThermoFisher EK0032 KAPA HiFi HS Uracil +ready Mix KAPA Biosystems KK2802 ATP (100 mM) Fermentas R0441 dNTP Mix (25 mM each) ThermoFisher R1121 Dynabeads MyOne Streptavidin C1 InvitrogenTM 65001 AccuPrime Pfx DNA polymerase ThermoFisher 12344024 T4 RNA ligase reaction buffer(10x) NEB B0216L FastAP thermosensitive alkaline phosphatase(1U/μL) ThermoFisher EF0651 T4 DNA ligase, 5 U/μL ThermoFisher EL0012 T4 DNA ligase, high concentrated 30 U/μL ThermoFisher EL0013 Klenow fragment (10 U/uL) ThermoFisher EP0052 PB buffer Qiagen 19066 AMPure XP Beads Beckman A63882 Isopropyl alcohol Macklin I811932-4L Anhydrous ethanol, pharmaceutical grade, 99.5% Mreda M206333-5L 5M NaCl Sigma-AldriCE S5150-1L Nuclease-free water Thermo Fisher 4387936 20× SSC buffer Sigma-AldriCE S6639-1L 3 M Sodium acetate, pH 5.2 Sigma-AldriCE S7899 Ethanol (Analytical Reagent) Sigma-AldriCE 459844 20% (wt/vol) SDS solution Thermo Scientific J63394.AK Tris (1 M)、pH 8.0、no RNase Thermo Scientific AM9856 Guanidine hydrochloride Sigma-Aldrich G3272-2 kg Water, HPLC grade Sigma-Aldrich AH365-4HC Proteinase K (10 mg/mL) Sigma-Aldrich P6556 0.5M EDTA (pH8.0) Applichen A48921000 NaClO Sinopharm 80010428 Agarose Biowest 111860 Critical commercial assays Qubit dsDNA Quantitation Kit Invitrogen Q32854 MinElute PCR Purification Kit QIAGEN 28006 Twist Mitochondrial Panel Kit Twist 102040 Twist Binding and Purification Beads Twist 100984 Twist Universal Blockers Twist 100767 Twist Hybridization Reagents Twist 100982 Twist Wash Buffers Twist 100846 Twist Ancient Human DNA Panel Twist 106658 Deposited data BAM files reported in this paper have been deposited in the GSA-Human (https://ngdc. cncb.ac.cn/gsa-human/). ..
Article Title: Cotranslational membrane protein biogenesis by an EMC-bound translocon.
Article Snippet: After adding Nature Structural & Molecular Biology Article https://doi.org/10.1038/s41594-026-01889-2 100 μl of chloroform (Sigma-Aldrich, cat. no. C2432), RNA in the aqueous layer was separated by centrifugation at 12,000g for 15 min at 4 °C and precipitated overnight by adding 0.1 volume of 3 M sodium acetate (Invitrogen, cat. no. AM9740), 20 μg of GlycoBlue (Invitrogen, cat. no. AM9515) and 1.2 volumes of isopropanol. .. Purified RNase footprints used for DNA library preparation were resuspended in 5 μl of T4 polynucleotide kinase (PNK) reaction mixture (1× PNK buffer, 10 U of PNK (NEB, cat. no. M0201L) and 10 U of SUPERaseIn (Invitrogen, cat. no. AM2694)) and end-repaired at 37 °C for 1 h. Next, 5 μl of polyadenylation mix containing 10 U of Escherichia coli poly (A) polymerase (NEB, cat. no. M0276L), 2 μl of 5× first-strand buffer (250 mM Tris-HCl pH 8.3, 375 mM KCl, 15 mM MgCl2), 0.25 mM ATP and 10 U SUPERaseIn was added, and the reaction was incubated at 37 °C for 2 h. The resulting polyadenylated RNAs were reverse transcribed by SMART-RT, by adding 2.5 μl of reverse transcription primer (10 μM, 5′-ACGTGTGCTCTTCCGATCTNNNNNN NNNNNNNNNTTTTTTTTTTTTTTTVN-3′, where ‘N’ is any base and ‘V’ is ‘A’, ‘C’ or ‘G’), annealing by heating to 72 °C for 5 min and cooling on ice, and then supplementing with 17.6 μl of SMART-RT mixture (2.5 μl of dNTP (10 mM), 400 U of Superscript II reverse transcriptase (Invitrogen, cat. no. 18064014), 10 U of SUPERaseIn, 4 μl of Superscript II first-strand buffer (5×), 1.5 μl of DTT (100 mM), 6 μl of Betaine (5 M), 0.12 μl of MgCl2 (1 M), 0.5 μl of template-switching oligos (100 μM 5′-CTCTTTCCCTACACGACGCTCTTCCGATCTNNNNrGrG+G-3′, where ‘N’ is any base and ‘+G’ is a locked nucleic acid guanylate) and 0.5 μl of nuclease-free water). ..
Binding Assay:Article Title: Reconstructing the west-east genetic division in Indonesia using ancient genomes
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Biological samples Ancient skeletal element This paper LMCM1 Ancient skeletal element This paper LMCM2 Chemicals, peptides, and recombinant proteins T4 Polynucleotide Kinase (10 U/uL) ThermoFisher EK0032 KAPA HiFi HS Uracil +ready Mix KAPA Biosystems KK2802 ATP (100 mM) Fermentas R0441 dNTP Mix (25 mM each) ThermoFisher R1121 Dynabeads MyOne Streptavidin C1 InvitrogenTM 65001 AccuPrime Pfx DNA polymerase ThermoFisher 12344024 T4 RNA ligase reaction buffer(10x) NEB B0216L FastAP thermosensitive alkaline phosphatase(1U/μL) ThermoFisher EF0651 T4 DNA ligase, 5 U/μL ThermoFisher EL0012 T4 DNA ligase, high concentrated 30 U/μL ThermoFisher EL0013 Klenow fragment (10 U/uL) ThermoFisher EP0052 PB buffer Qiagen 19066 AMPure XP Beads Beckman A63882 Isopropyl alcohol Macklin I811932-4L Anhydrous ethanol, pharmaceutical grade, 99.5% Mreda M206333-5L 5M NaCl Sigma-AldriCE S5150-1L Nuclease-free water Thermo Fisher 4387936 20× SSC buffer Sigma-AldriCE S6639-1L 3 M Sodium acetate, pH 5.2 Sigma-AldriCE S7899 Ethanol (Analytical Reagent) Sigma-AldriCE 459844 20% (wt/vol) SDS solution Thermo Scientific J63394.AK Tris (1 M)、pH 8.0、no RNase Thermo Scientific AM9856 Guanidine hydrochloride Sigma-Aldrich G3272-2 kg Water, HPLC grade Sigma-Aldrich AH365-4HC Proteinase K (10 mg/mL) Sigma-Aldrich P6556 0.5M EDTA (pH8.0) Applichen A48921000 NaClO Sinopharm 80010428 Agarose Biowest 111860 Critical commercial assays Qubit dsDNA Quantitation Kit Invitrogen Q32854 MinElute PCR Purification Kit QIAGEN 28006 Twist Mitochondrial Panel Kit Twist 102040 Twist Binding and Purification Beads Twist 100984 Twist Universal Blockers Twist 100767 Twist Hybridization Reagents Twist 100982 Twist Wash Buffers Twist 100846 Twist Ancient Human DNA Panel Twist 106658 Deposited data BAM files reported in this paper have been deposited in the GSA-Human (https://ngdc. cncb.ac.cn/gsa-human/). ..
Hybridization:Article Title: Reconstructing the west-east genetic division in Indonesia using ancient genomes
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Biological samples Ancient skeletal element This paper LMCM1 Ancient skeletal element This paper LMCM2 Chemicals, peptides, and recombinant proteins T4 Polynucleotide Kinase (10 U/uL) ThermoFisher EK0032 KAPA HiFi HS Uracil +ready Mix KAPA Biosystems KK2802 ATP (100 mM) Fermentas R0441 dNTP Mix (25 mM each) ThermoFisher R1121 Dynabeads MyOne Streptavidin C1 InvitrogenTM 65001 AccuPrime Pfx DNA polymerase ThermoFisher 12344024 T4 RNA ligase reaction buffer(10x) NEB B0216L FastAP thermosensitive alkaline phosphatase(1U/μL) ThermoFisher EF0651 T4 DNA ligase, 5 U/μL ThermoFisher EL0012 T4 DNA ligase, high concentrated 30 U/μL ThermoFisher EL0013 Klenow fragment (10 U/uL) ThermoFisher EP0052 PB buffer Qiagen 19066 AMPure XP Beads Beckman A63882 Isopropyl alcohol Macklin I811932-4L Anhydrous ethanol, pharmaceutical grade, 99.5% Mreda M206333-5L 5M NaCl Sigma-AldriCE S5150-1L Nuclease-free water Thermo Fisher 4387936 20× SSC buffer Sigma-AldriCE S6639-1L 3 M Sodium acetate, pH 5.2 Sigma-AldriCE S7899 Ethanol (Analytical Reagent) Sigma-AldriCE 459844 20% (wt/vol) SDS solution Thermo Scientific J63394.AK Tris (1 M)、pH 8.0、no RNase Thermo Scientific AM9856 Guanidine hydrochloride Sigma-Aldrich G3272-2 kg Water, HPLC grade Sigma-Aldrich AH365-4HC Proteinase K (10 mg/mL) Sigma-Aldrich P6556 0.5M EDTA (pH8.0) Applichen A48921000 NaClO Sinopharm 80010428 Agarose Biowest 111860 Critical commercial assays Qubit dsDNA Quantitation Kit Invitrogen Q32854 MinElute PCR Purification Kit QIAGEN 28006 Twist Mitochondrial Panel Kit Twist 102040 Twist Binding and Purification Beads Twist 100984 Twist Universal Blockers Twist 100767 Twist Hybridization Reagents Twist 100982 Twist Wash Buffers Twist 100846 Twist Ancient Human DNA Panel Twist 106658 Deposited data BAM files reported in this paper have been deposited in the GSA-Human (https://ngdc. cncb.ac.cn/gsa-human/). ..
Sequencing:
Generated:
Ligation:Article Title: PUS7 pseudouridylation of 7SK orchestrates human somatic cell reprogramming into iPS cells.
Article Snippet: .. For RT and splint ligation, 1 μl of 10× annealing buffer (250 mM Tris-HCl pH 7.4 and 480 mM KCl) and 1 μl of 0.5 μM RT primer (5′-CCA CAT GCA GCG CCT CAT TTG G-3′) were added and the mixture was heated to 93 °C for 2 min. Then, 5 μl of 2× AMV reverse transcriptase reaction mix was added and the RT reaction was performed at 42 °C for 1 h, followed by 85 °C for 5 min. To degrade RNA, 1 μl of RNase H (New England Biolabs, M0297L) was added and incubated at 37 °C for 20 min and then at 85 °C for 5 min. For splint ligation, 1 μl of 1.5 μM adaptor/splint oligo mix (adaptor, 5′-/5Phos/CCA TGG ACC GAA GAC CGG TCC TCC TCT A-3′; splint, 5′-CGG TCC ATG GAG GAG AAC GT/3SpC3/-3′) was added and heated to 75 °C for 3 min. .. Subsequently, T4 DNA ligase (New England Biolabs, M0202L) and 1× ligase buffer with 12.5% DMSO were added and ligation was performed at 16 °C for 16 h, followed by heat inactivation at 65 °C for 10 min. Lastly, 2 μl of the reaction mixture was used as a template for PCR amplification (35 cycles) using the forward primer 5′-TAG AGG AGG ACC GGT CTT CGG TC-3′ and the RT primer as the reverse primer.
Chloramphenicol Acetyltransferase Assay:Article Title: PUS7 pseudouridylation of 7SK orchestrates human somatic cell reprogramming into iPS cells.
Article Snippet: .. For RT and splint ligation, 1 μl of 10× annealing buffer (250 mM Tris-HCl pH 7.4 and 480 mM KCl) and 1 μl of 0.5 μM RT primer (5′-CCA CAT GCA GCG CCT CAT TTG G-3′) were added and the mixture was heated to 93 °C for 2 min. Then, 5 μl of 2× AMV reverse transcriptase reaction mix was added and the RT reaction was performed at 42 °C for 1 h, followed by 85 °C for 5 min. To degrade RNA, 1 μl of RNase H (New England Biolabs, M0297L) was added and incubated at 37 °C for 20 min and then at 85 °C for 5 min. For splint ligation, 1 μl of 1.5 μM adaptor/splint oligo mix (adaptor, 5′-/5Phos/CCA TGG ACC GAA GAC CGG TCC TCC TCT A-3′; splint, 5′-CGG TCC ATG GAG GAG AAC GT/3SpC3/-3′) was added and heated to 75 °C for 3 min. .. Subsequently, T4 DNA ligase (New England Biolabs, M0202L) and 1× ligase buffer with 12.5% DMSO were added and ligation was performed at 16 °C for 16 h, followed by heat inactivation at 65 °C for 10 min. Lastly, 2 μl of the reaction mixture was used as a template for PCR amplification (35 cycles) using the forward primer 5′-TAG AGG AGG ACC GGT CTT CGG TC-3′ and the RT primer as the reverse primer.
Incubation:Article Title: PUS7 pseudouridylation of 7SK orchestrates human somatic cell reprogramming into iPS cells.
Article Snippet: .. For RT and splint ligation, 1 μl of 10× annealing buffer (250 mM Tris-HCl pH 7.4 and 480 mM KCl) and 1 μl of 0.5 μM RT primer (5′-CCA CAT GCA GCG CCT CAT TTG G-3′) were added and the mixture was heated to 93 °C for 2 min. Then, 5 μl of 2× AMV reverse transcriptase reaction mix was added and the RT reaction was performed at 42 °C for 1 h, followed by 85 °C for 5 min. To degrade RNA, 1 μl of RNase H (New England Biolabs, M0297L) was added and incubated at 37 °C for 20 min and then at 85 °C for 5 min. For splint ligation, 1 μl of 1.5 μM adaptor/splint oligo mix (adaptor, 5′-/5Phos/CCA TGG ACC GAA GAC CGG TCC TCC TCT A-3′; splint, 5′-CGG TCC ATG GAG GAG AAC GT/3SpC3/-3′) was added and heated to 75 °C for 3 min. .. Subsequently, T4 DNA ligase (New England Biolabs, M0202L) and 1× ligase buffer with 12.5% DMSO were added and ligation was performed at 16 °C for 16 h, followed by heat inactivation at 65 °C for 10 min. Lastly, 2 μl of the reaction mixture was used as a template for PCR amplification (35 cycles) using the forward primer 5′-TAG AGG AGG ACC GGT CTT CGG TC-3′ and the RT primer as the reverse primer.
Article Title: Cotranslational membrane protein biogenesis by an EMC-bound translocon.
Article Snippet: After adding Nature Structural & Molecular Biology Article https://doi.org/10.1038/s41594-026-01889-2 100 μl of chloroform (Sigma-Aldrich, cat. no. C2432), RNA in the aqueous layer was separated by centrifugation at 12,000g for 15 min at 4 °C and precipitated overnight by adding 0.1 volume of 3 M sodium acetate (Invitrogen, cat. no. AM9740), 20 μg of GlycoBlue (Invitrogen, cat. no. AM9515) and 1.2 volumes of isopropanol. .. Purified RNase footprints used for DNA library preparation were resuspended in 5 μl of T4 polynucleotide kinase (PNK) reaction mixture (1× PNK buffer, 10 U of PNK (NEB, cat. no. M0201L) and 10 U of SUPERaseIn (Invitrogen, cat. no. AM2694)) and end-repaired at 37 °C for 1 h. Next, 5 μl of polyadenylation mix containing 10 U of Escherichia coli poly (A) polymerase (NEB, cat. no. M0276L), 2 μl of 5× first-strand buffer (250 mM Tris-HCl pH 8.3, 375 mM KCl, 15 mM MgCl2), 0.25 mM ATP and 10 U SUPERaseIn was added, and the reaction was incubated at 37 °C for 2 h. The resulting polyadenylated RNAs were reverse transcribed by SMART-RT, by adding 2.5 μl of reverse transcription primer (10 μM, 5′-ACGTGTGCTCTTCCGATCTNNNNNN NNNNNNNNNTTTTTTTTTTTTTTTVN-3′, where ‘N’ is any base and ‘V’ is ‘A’, ‘C’ or ‘G’), annealing by heating to 72 °C for 5 min and cooling on ice, and then supplementing with 17.6 μl of SMART-RT mixture (2.5 μl of dNTP (10 mM), 400 U of Superscript II reverse transcriptase (Invitrogen, cat. no. 18064014), 10 U of SUPERaseIn, 4 μl of Superscript II first-strand buffer (5×), 1.5 μl of DTT (100 mM), 6 μl of Betaine (5 M), 0.12 μl of MgCl2 (1 M), 0.5 μl of template-switching oligos (100 μM 5′-CTCTTTCCCTACACGACGCTCTTCCGATCTNNNNrGrG+G-3′, where ‘N’ is any base and ‘+G’ is a locked nucleic acid guanylate) and 0.5 μl of nuclease-free water). ..
other:Article Title: AAV treatment of Huntington's disease
Article Snippet: Sections were stained for 2 minutes with diaminobenzidine using the Metal Enhanced DAB Substrate Kit (Pierce).
DNA Library Preparation:Article Title: Cotranslational membrane protein biogenesis by an EMC-bound translocon.
Article Snippet: After adding Nature Structural & Molecular Biology Article https://doi.org/10.1038/s41594-026-01889-2 100 μl of chloroform (Sigma-Aldrich, cat. no. C2432), RNA in the aqueous layer was separated by centrifugation at 12,000g for 15 min at 4 °C and precipitated overnight by adding 0.1 volume of 3 M sodium acetate (Invitrogen, cat. no. AM9740), 20 μg of GlycoBlue (Invitrogen, cat. no. AM9515) and 1.2 volumes of isopropanol. .. Purified RNase footprints used for DNA library preparation were resuspended in 5 μl of T4 polynucleotide kinase (PNK) reaction mixture (1× PNK buffer, 10 U of PNK (NEB, cat. no. M0201L) and 10 U of SUPERaseIn (Invitrogen, cat. no. AM2694)) and end-repaired at 37 °C for 1 h. Next, 5 μl of polyadenylation mix containing 10 U of Escherichia coli poly (A) polymerase (NEB, cat. no. M0276L), 2 μl of 5× first-strand buffer (250 mM Tris-HCl pH 8.3, 375 mM KCl, 15 mM MgCl2), 0.25 mM ATP and 10 U SUPERaseIn was added, and the reaction was incubated at 37 °C for 2 h. The resulting polyadenylated RNAs were reverse transcribed by SMART-RT, by adding 2.5 μl of reverse transcription primer (10 μM, 5′-ACGTGTGCTCTTCCGATCTNNNNNN NNNNNNNNNTTTTTTTTTTTTTTTVN-3′, where ‘N’ is any base and ‘V’ is ‘A’, ‘C’ or ‘G’), annealing by heating to 72 °C for 5 min and cooling on ice, and then supplementing with 17.6 μl of SMART-RT mixture (2.5 μl of dNTP (10 mM), 400 U of Superscript II reverse transcriptase (Invitrogen, cat. no. 18064014), 10 U of SUPERaseIn, 4 μl of Superscript II first-strand buffer (5×), 1.5 μl of DTT (100 mM), 6 μl of Betaine (5 M), 0.12 μl of MgCl2 (1 M), 0.5 μl of template-switching oligos (100 μM 5′-CTCTTTCCCTACACGACGCTCTTCCGATCTNNNNrGrG+G-3′, where ‘N’ is any base and ‘+G’ is a locked nucleic acid guanylate) and 0.5 μl of nuclease-free water). ..
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