Human lung proteins can advance or thwart SARS-CoV-2 infections NatureGenet
was calculated from the distribution of the negative log MaGeCK scores in the relevant top 100 gene set.Lentivirus for Cas9 KO was produced in six-well plates. In brief, low-passage HEK293FT cells were grown in DMEM supplemented with 10% FBS and passaged using TrypLE . For viral plasmid transfection, polyethylenimine ‘Max’ at a concentration of 1 mg mland pH 7.1 was used. PEI was mixed with 1 ml of DMEM. pMD2.G, PAX and target-guide plasmid .
CoV-2 used was obtained from BEI Resources. The original stock from BEI was passaged through a 0.45 µM syringe filter and then 5 µl was inoculated onto 80% confluent T175 flasks of Vero E6 cells to produce our p1 stock. The CPE was monitored daily, and flasks were frozen when cells exhibited ~70% CPE, ~48–72 h postinfection. Lysates were then thawed, collected and cell debris was spun down at 3,000 r.p.m. for 20 min.
CoV-2 variant viral stocks were produced as described previously. In brief, the B.1.351 isolate was derived from a nasal swab at Stanford Hospital. Nasal swab medium was inoculated onto Vero E6 cells in the BSL-3 facility at Stanford University. Vero E6 cells were monitored for CPE and harvested as above for p0 stocks. p1 and p2 stocks were generated as above. Evaluation of antiviral activity of chemical compounds was conducted as described previously.
CoV-2 at an MOI of 0.05 and TCIDwas determined on whole-cell lysates 24 h postinfection. To further confirm that our viral stocks were responsive to furin cleavage, we also infected HPMEC/ACE2 , with
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