Journal: Molecular cell

Article Title: Transcription of nearly all yeast RNA Polymerase II-transcribed genes is dependent on transcription factor TFIID

doi: 10.1016/j.molcel.2017.08.014

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-V5 Invitrogen Cat # 46-0705 Anti-FLAG M2 Magnetic Beads Sigma-Aldrich Cat # M8823 Rabbit polyclonal anti-Taf1 Hahn lab Rabbit # 3506 Rabbit polyclonal anti-Taf4 Hahn lab Rabbit # 4022 Rabbit polyclonal anti-Taf12 Hahn lab Rabbit # 3434 Rabbit polyclonal anti-Taf3 Hahn lab Rabbit # 4045 Rabbit polyclonal anti-TBP Hahn lab Rabbit # 7370 Rabbit polyclonal anti-H3 Abcam Cat # ab1791 Rabbit polyclonal anti-Taf4 P. Anthony Weil N/A Rabbit polyclonal anti-Taf5 P. Anthony Weil N/A Rabbit polyclonal anti-Taf11 P. Anthony Weil N/A Biological Samples Chemicals, Peptides, and Recombinant Proteins 3-Indoleacetic acid (3-IAA) Sigma-Aldrich I3750; CAS: 87-51-4 Micrococcal Nuclease (MNase) Worthington Biochemical Corporation Cat # NFCB cOmplete Mini, EDTA-free Protease inhibitor cocktail tablets Roche Cat # 11836170001 4-thiouracil Sigma-Aldrich Cat # 440736; CAS: 591-28-6 MTSEA biotin-XX Biotium Cat # 90066 Dynabeads MyOne Streptavidin C1 Invitrogen, ThermoFisher Cat # 65001 AMPureXP beads Beckman Coulter Cat # A63881 EZ-Link HPDP Biotin ThermoFisher Cat # 21341 Rapamycin Euromedex Cat # SYN-1185 Dyanabeads Protein G Thermo Fisher Scientific Cat # 10004D 0.5 mm zirconia disruption beads Research Products International Cat # 9834 Critical Commercial Assays RiboPure yeast kit Ambion, Life Technologies Cat # AM1926 RNeasy MinElute Cleanup Kit Qiagen Cat # 742040 μMACS Streptavidin Kit Miltenyi Biotec Cat# 130-074-101 Deposited Data All sequencing datasets have been uploaded in GEO under accession GSE97081 Western blot original images Mendeley Data file http://dx.doi.org/10.17632/gwtwc4ndpj.1 Experimental Models: Cell Lines Experimental Models: Organisms/Strains S. pombe : Strain background: 972h Laboratory of Gerald Smith ATCC: 24843 S. cerevisiae : Strain background: BY4705 Laboratory of Dan Gottschling ATCC: 200869 Yeast strains, see Table S2 This paper N/A Recombinant DNA Sequence-Based Reagents Primers for RT-qPCR, see Table S1 This paper N/A Software and Algorithms Bowtie2 http://bowtie-bio.sourceforge.net/bowtie2/index.shtml N/A Shell Scripts for Sequence Alignment This paper Perl Scripts for analysis This paper R Scripts for analysis This paper Other Open in a separate window KEY RESOURCES TABLE

Techniques: Magnetic Beads, Recombinant, Protease Inhibitor, Sequencing, Western Blot, Software

Journal: bioRxiv

Article Title: Expression of human ACE2 N-terminal domain, part of the receptor for SARS-CoV-2, in fusion with maltose binding protein, E. coli ribonuclease I and human RNase A

doi: 10.1101/2021.01.31.429007

Figure Lengend Snippet: A. A schematic diagram of MBP-RNase A fusion. B. Total cellular proteins from IPTG-induced cells (NEB Express). C. Supernatant (soluble) of the fusion proteins (3 independent isolates each). Arrows indicate the fusions. D. Comparison of supernatant (Sol.) and pellet (Inc.) of the fusions. M, protein size marker (NEB). E. SDS-PAGE analysis of partially purified MBP-RNase I fusion. Lane 1, protein size marker; lane 2, MBP-RNase I fusion purified from IPTG-induced NEB Turbo cells; lane 3, RNase I f (NEB). F. RNase activity on Fluorescein-labeled RNA (300 nt). No enzyme (uncut) and MBP-ACE2NTD fusion serve as negative controls.

Article Snippet: The protein yield of MBP-RNase I was highest in NEB Turbo cells, probably due to the robust cell growth and higher cell weight per liter culture. shows the partially purified MBP-RNase I fusion in comparison with RNase I f , (RNase I f = MBP-RNase I fusion from NEB as a research grade ribonuclease, M0243S).

Techniques: Marker, SDS Page, Purification, Activity Assay, Labeling

Journal: bioRxiv

Article Title: Expression of human ACE2 N-terminal domain, part of the receptor for SARS-CoV-2, in fusion with maltose binding protein, E. coli ribonuclease I and human RNase A

doi: 10.1101/2021.01.31.429007

Figure Lengend Snippet: MBP-ACE2NTD (ACE), MBP-TMPRSS2 (PRS, lacking the transmembrane domain), MBP-RNase I (RI), MBP-RNase A (RA). A. SDS-PAGE analysis of total proteins in cell lysate. B. SDS-PAGE analysis of soluble proteins (supernatant) in cell lysate. “*” indicates the expected target protein.

Article Snippet: The protein yield of MBP-RNase I was highest in NEB Turbo cells, probably due to the robust cell growth and higher cell weight per liter culture. shows the partially purified MBP-RNase I fusion in comparison with RNase I f , (RNase I f = MBP-RNase I fusion from NEB as a research grade ribonuclease, M0243S).

Techniques: SDS Page

Journal: bioRxiv

Article Title: Expression of human ACE2 N-terminal domain, part of the receptor for SARS-CoV-2, in fusion with maltose binding protein, E. coli ribonuclease I and human RNase A

doi: 10.1101/2021.01.31.429007

Figure Lengend Snippet: A. MBP-RNase I ribonuclease activity assay. The low range ssRNA ladder (50 to 1000 nt long, NEB) was used as the substrate for RNase activity assay in a high sale buffer (100 mM NaCl, 50 mM Tris-HCl, pH 7.5) supplemented with divalent cations (1 mM) or EDTA (10 mM).

Article Snippet: The protein yield of MBP-RNase I was highest in NEB Turbo cells, probably due to the robust cell growth and higher cell weight per liter culture. shows the partially purified MBP-RNase I fusion in comparison with RNase I f , (RNase I f = MBP-RNase I fusion from NEB as a research grade ribonuclease, M0243S).

Techniques: Activity Assay

Journal: bioRxiv

Article Title: Expression of human ACE2 N-terminal domain, part of the receptor for SARS-CoV-2, in fusion with maltose binding protein, E. coli ribonuclease I and human RNase A

doi: 10.1101/2021.01.31.429007

Figure Lengend Snippet: A. Purification of RNase I (6xHis) from Nickel-NTA agarose column. Lane 1, RNase I (6xHis) pooled fractions from a nickel column (purified from T7 Express cell extract). Arrows indicate the cytoplasmic RNase I precursor (cRNase I) with the signal peptide (predicted MW 30.7 kDa), and the periplasmic RNase I with the signal peptide removed (predicted MW 27.0 kDa). B. RNase activity on a FAM-labeled COVID-19 RNA (60 mer). S = substrate; P = cleavage product(s). Positive controls, 50 and 5 U of RNase I f (MBP-RNase I fusion, NEB). RNase I (6xHis) enzyme titration (2 μg to 25 ng protein) was used in the activity assay to digest fixed amount of RNA (16 nM) in NEB buffer 3 at 37°C for 1 h. Proteinase K (1.6 U) was added to remove RNase I. The final cleavage products were analyzed by capillary electrophoresis (CE) and peaks were visualized by PeakScan.

Article Snippet: The protein yield of MBP-RNase I was highest in NEB Turbo cells, probably due to the robust cell growth and higher cell weight per liter culture. shows the partially purified MBP-RNase I fusion in comparison with RNase I f , (RNase I f = MBP-RNase I fusion from NEB as a research grade ribonuclease, M0243S).

Techniques: Purification, Nickel Column, Activity Assay, Labeling, Titration, Electrophoresis

Journal: bioRxiv

Article Title: Expression of human ACE2 N-terminal domain, part of the receptor for SARS-CoV-2, in fusion with maltose binding protein, E. coli ribonuclease I and human RNase A

doi: 10.1101/2021.01.31.429007

Figure Lengend Snippet: A. Schematic diagram of RNase I-ACENTD (6xHis) fusion. B. Western blot analysis of RNase I-ACE2NTD in total protein, supernatant (soluble), and refolded protein using anti-6xHis Ab. C. Same as in B , except using anti-ACE2 monoclonal Ab. D. SDS-PAGE analysis of the refolded RNase I-ACE2NTD fusion and further purified protein by Ni magnetic beads and Ni spin column. E. RNase I-ACE2NTD (refolded) ribonuclease activity on Fluorescein (FL)-labeled DNA (300 nt) in NEB buffer 3. RNase I (6xHis) and MBP-RNase I were used as positive controls. F. Ribonuclease activity of RNase I-ACE2NTD (purified by Ni magnetic beads or Ni spin column) on COVID-19 RNA (60mer). RNase I f , a positive control. FAM-S, FAM-labeled substrate; FAM-P, FAM labeled cleavage product(s).

Article Snippet: The protein yield of MBP-RNase I was highest in NEB Turbo cells, probably due to the robust cell growth and higher cell weight per liter culture. shows the partially purified MBP-RNase I fusion in comparison with RNase I f , (RNase I f = MBP-RNase I fusion from NEB as a research grade ribonuclease, M0243S).

Techniques: Western Blot, SDS Page, Purification, Magnetic Beads, Activity Assay, Labeling, Positive Control