Journal: Research Square
Article Title: Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs
Figure Lengend Snippet: Calu-3-Cas9 cells were stably transduced to express 2 different sgRNAs (g1, g2) per indicated gene and selected for 10–15 days. a. Cells were infected with SARS-CoV-2 bearing the mNG reporter and the infection efficiency was scored 48h later by flow cytometry. b. The expression levels of ACE2 were analyzed by immunoblot, Actin served as a loading control. c. Relative surface ACE2 expression was measured using a Spike-RBD-Fc fusion and a fluorescent secondary antibody followed by flow cytometry analysis. d. Cells were incubated with SARS-CoV-2 at MOI 5 for 2h at 37°C and then treated with Subtilisin A followed by RNA extraction and RdRp RT-qPCR analysis as a measure of viral internalization. e. Cells were infected with Spike del19 and VSV-G pseudotyped, GFP expressing VSV and infection efficiency was analyzed 24h later by flow cytometry. f. Cells were infected with SARS-CoV-2 at MOI 0.05 and, 24h later, lysed for RNA extraction and RdRp RT-qPCR analysis. g. Aliquots of the supernatants from F were harvested and plaque assays were performed to evaluate the production of infectious viruses in the different conditions. h. Cells were infected with MERS-CoV and 16h later, infectious particle production in the supernatant was measured by TCID 50 . The mean and SEM of at least 5 ( a ), 3 ( c , d , e , f , h ) independent experiments or representative experiments ( b and g ) are shown. The red dashed line represents 50% inhibition ( a , c-f ).
Article Snippet: Cells were lysed in lysis buffer (10 mM TRIS 1M pH7.6, NaCl 150 mM, Triton X100 1%, EDTA 1 mM, deoxycholate 0,1%) supplemented with sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 5% glycerol, 100 mM DTT, 0.02% bromophenol blue), resolved by SDS-PAGE and analyzed by immunoblotting using primary antibodies against ACE2 (ProteinTech 21115–1-P) and Actin (Sigma-Aldrich A1978), followed by HRP-conjugated anti-rabbit or anti-mouse immunoglobulin antibodies and chemiluminescence Clarity or Clarity max substrate (Bio-Rad).
Techniques: Stable Transfection, Infection, Flow Cytometry, Expressing, Western Blot, Incubation, RNA Extraction, Quantitative RT-PCR, Inhibition