Journal: Bio-protocol

Article Title: QsRNA-seq: A protocol for generating libraries for high-throughput sequencing of small RNAs

doi: 10.21769/BioProtoc.3179

Figure Lengend Snippet:

Article Snippet: RNase-free plastic pipette tips with filter (10 μl, 20 μl, 200 μl, 1,000 μl) 1.7 ml RNase-free microtubes 0.2 ml RNase-free PCR tubes miRVana miRNA Isolation Kit (Thermo Fisher scientific, catalog number: AM1560) or TRIzol reagent (Ambion, catalog number: 15596026) with Direct-Zol RNA MiniPrep Plus (Zymo Research, catalog number: R2072) RiboLock RNase Inhibitor (Thermo Fisher Scientific, catalog number: EO0381) T4 RNA ligase 1 (ssRNA Ligase) (NEB, catalog number: M0204S) T4 RNA ligase 2, truncated (NEB, catalog number: M0242S) RppH enzyme (NEB, catalog number M0356S) QScript Flex cDNA synthesis kit (Quanta, catalog number: 95049-025) Phusion High-Fidelity DNA Polymerase (NEB, catalog number: M0530S) Agencourt Ampure XP (Beckman Coulter, catalog number: A63881) or SPRI select reagent Kit (Beckman Coulter, catalog number: {"type":"entrez-nucleotide","attrs":{"text":"B23319","term_id":"2508950","term_text":"B23319"}} B23319 ) RNA BR assay kit (Quibit, catalog number: {"type":"entrez-protein","attrs":{"text":"Q10210","term_id":"1723278","term_text":"Q10210"}} Q10210 ) dsDNA HS assay kit (Quibit, catalog number: {"type":"entrez-protein","attrs":{"text":"Q10210","term_id":"1723278","term_text":"Q10210"}} Q10210 ) PEG 8000 (NEB, catalog number: B1004) Nuclease-free water (Sigma-Aldrich, catalog number: W4502) Ethyl Alcohol absolute Isopropyl-alcohol (Isopropanol) chemical grade Ethidium Bromide (hylabs, catalog number: BP451) Gel loading dye purple (6x) (NEB, catalog number: B7024) [Optional] Low-range ultra agarose (Bio-Rad, catalog number: 161-3107) ATP (NEB, catalog number: P0756S) DMSO (NEB, catalog number B0515) Tris (Spectrum, catalog number: s1519) Boric Acid (Bio-Lab, catalog number: 000201059100) EDTA (J.T.

Techniques:

Journal: Cell

Article Title: Structure of the MIND Complex Defines a Regulatory Focus for Yeast Kinetochore Assembly

doi: 10.1016/j.cell.2016.10.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, Peptides, and Recombinant Proteins Dsn1 560-572 QQLLKGLSLSFSK Tufts University Core Facility N/A FITC-Dsn1 560-572 FITC-AHA-QQLLKGLSLSFSK Tufts University Core Facility N/A Mtw1 230-262 KDFRTRYIDIRTNNVLRKLGLLGDKEDEKQSAK Tufts University Core Facility N/A FITC-Mtw1 230-262 FITC-AHA-KDFRTRYIDIRTNNVLRKLGLLGDKEDEKQSAK Tufts University Core Facility N/A Mtw1 274-289 SIDIEEPQLDLLDDVL Tufts University Core Facility N/A FITC-Mtw1 274-289 FITC-AHA- SIDIEEPQLDLLDDVL Tufts University Core Facility N/A FITC-Mif2 1-41 MDYMNLGVKSRKTGLTVNKTVQKDEYSMENLNDFFKDEQDS Tufts University Core Facility N/A FITC-Ame1 1-25 MDALKQRHLKLLYRQRGSASRTIDY Tufts University Core Facility N/A PEG 550 MME Hampton Research HR2-611 PGA-LM Molecular Dimensions MD2-250-108 Ethylmercurithiosalicylic acid, sodium salt Heavy Atom Screen M1 Hampton Research HR2-448 L(+)-Selenomethionine Acros Organics 3211-76-5 PEG 8000 Sigma 81268-1kg Tantalum Cluster Derivatization Kit Jena Bioscience PK-103 PEG 3000 Rigaku 1008057 Deposited Data Atomic coordinates, MIND-C1 structure Protein Data Bank PDB: 5T58 Atomic coordinates, MN-C2 structure Protein Data Bank PDB: 5T51 Atomic coordinates, MN-C2-Mif2 1-41 structure Protein Data Bank PDB: 5T59 Atomic coordinates, Spc24/Spc25-Dsn1 560-572 structure Protein Data Bank PDB: 5T6J Experimental Models: Organisms/Strains E. coli Rosetta 2 (DE3) EMD #71400 Recombinant DNA pLIC_M42 H 6 -TEV- *MIND-C1 This study N/A pLIC_M147 H 6 -TEV- *MIND-C1:D205 This study N/A pLIC_M147_mut2 H 6 -TEV- *MIND-C1:D205-2D This study N/A pLIC_M142 H 6 -TEV- *MIND-C1:D367 This study N/A pLIC_M89 H 6 -TEV- *head I MN-C2 This study N/A pLIC_M89_mut4 H 6 -TEV- *head I Site II mut.

Techniques: Recombinant, Software

Journal: Molecular and biochemical parasitology

Article Title: A unified approach towards Trypanosoma brucei functional genomics using Gibson assembly

doi: 10.1016/j.molbiopara.2016.08.001

Figure Lengend Snippet: [A] Schematic of the plasmid, showing the 5′ UTR targeting segment (1; 5′ UTR), a segment (3) containing the blasticidin selection marker (BSD), alpha/beta tubulin intergenic rection (INTER), triple-Ty1 tag (3X Ty1), the first 500 bp of FC1 (2; FC1 Cod). and the endogenous tagging vector backbone for inducible expression (ET vector, 4). Sizes for each DNA segment are shown in parentheses. [B] Agarose gel showing the DNA segments used for Gibson assembly (1–4, as labeled in A), and the product (P) of the assembly digested with PacI and NsiI to show the completed endogenous tagging insert. The asterisk in lane 3 denotes the BLA-INTER-3X Ty1 DNA segment, which was excised from a previously-assembled construct by BamHI and HindIII restriction digest. The asterisk in lane 4 denotes the ET plasmid backbone, which was isolated by PacI and NsiI digest. [C] Cells containing the FC1 endogenous tagging construct were fixed and stained with an antibody against the flagella connector (FC; red), anti-Ty1 (Ty1-FC1; green), and DAPI to label the DNA (DNA; blue). Cells were imaged by brightfield and fluorescence microscopy. Scale bar is 5 μm.

Article Snippet: The homemade Master Mix was prepared by combining 699 μL water, 320 μL 5x isothermal reaction buffer (500 mM Tris-Cl, pH 7.5, 250 mg/mL PEG-8000, 50 mM MgCl 2 , 50 mM DTT, 1 mM each of four dNTPs, 5 mM beta-NAD), 0.64 μL T5 Exonuclease (Epicentre, 10 U/μL), 20 μL Phusion DNA polymerase (NEB, 2 U/μL) and 160 μL Taq DNA ligase (NEB, 40 U/μL).

Techniques: Plasmid Preparation, Selection, Marker, Expressing, Agarose Gel Electrophoresis, Labeling, Construct, Isolation, Staining, Fluorescence, Microscopy

Journal: Molecular and biochemical parasitology

Article Title: A unified approach towards Trypanosoma brucei functional genomics using Gibson assembly

doi: 10.1016/j.molbiopara.2016.08.001

Figure Lengend Snippet: [A] Schematic of the plasmid, showing the last 500 bp of the 4400 gene, which functions as a targeting segment (1; 4400 Cod), the triple-Ty1 tag (2; 3X Ty1), the alpha/beta tubulin intergenic region (3; INTER), the puromycin resistance gene (4; PAC), a 500 bp segment of the 3′ UTR of 4400 used for targeting (5; 3′ UTR), and the endogenous tagging vector backbone for inducible expression (ET vector, 6). Sizes for each DNA segment are shown in parentheses. [B] Agarose gel showing the DNA segments used for Gibson assembly (1–6, as labeled in A), and the product (P) of the assembly digested with PacI and NsiI to show the tagged insert. The asterisk in lane 6 denotes the plasmid backbone of the ET vector, which was used for the Gibson reaction. [C] Cells containing the 4400 endogenous tagging construct were fixed and stained with an antibody that detects the basal body and bilobe structure (BB + Bilobe; red), anti-Ty1 (Ty1-FC1; green), and DAPI to label the DNA (DNA; blue). Cells were imaged by brightfield and fluorescence microscopy. Scale bar is 5 μm.

Article Snippet: The homemade Master Mix was prepared by combining 699 μL water, 320 μL 5x isothermal reaction buffer (500 mM Tris-Cl, pH 7.5, 250 mg/mL PEG-8000, 50 mM MgCl 2 , 50 mM DTT, 1 mM each of four dNTPs, 5 mM beta-NAD), 0.64 μL T5 Exonuclease (Epicentre, 10 U/μL), 20 μL Phusion DNA polymerase (NEB, 2 U/μL) and 160 μL Taq DNA ligase (NEB, 40 U/μL).

Techniques: Plasmid Preparation, Expressing, Agarose Gel Electrophoresis, Labeling, Construct, Staining, Fluorescence, Microscopy