NextSeq 500 sequencing; CRISPR screen of human embryonic stem cell line h-pES10 for the identification of host factors for SARS-COV-2 infection
Cell culture and maintainance: For the genetic screen the haploid hESC line, h-pES10, was used to generate a genome-wide loss-of-function library. The mutant haploid hESC library was cultured at 37 °C and 5% CO2 in feeder-free conditions on matrigel-coated plates (Corning) in mTeSR1 (STEMCELL Technologies) supplemented with 10 μM ROCK inhibitor Y-27632 (Stemgent) for 1 day after splitting. Before reaching confluency, cells were passaged by a quick trypsinization using TrypLE Select (Thermo Fisher Scientific). Vero E6 cells were maintained in Eagle's Minimum Essential Medium (EMEM; Biological Industries, Beit Haemek, Israel), supplemented with 10% fetal bovine serum, 2 mM L-Glutamine, 10 IU/ml Penicillin, and 10 μg/ml streptomycin (Biological Industries, Beit Haemek, Israel). Definitive endoderm differentiation assay: Mutant h-pES10 library cells were used for differentiation into definitive endoderm (DE). Cells were plated on Matrigel-coated 6-well plates at a density of 2 million cells per well. The next day, STEMdiff™ Definitive Endoderm Kit (STEMCELL Technologies, cat. no. 05110) has been used according to manufacturer's instructions. Briefly, the differentiation medium (2 ml/well) with its appropriate supplements was replaced every 24 hours for 4 days. SARS-CoV-2 Screen: On the 5th day of differentiation, differentiated DE cells were infected with SARS-CoV-2 at various MOIs. Following infection, cells were continued to be cultured in the DE medium for additional 5 days. Cells were allowed to undergo viral cytotoxicity and harvested 5 days post-infection for further analysis. 2 screen samples were infected with SARS-COV-2, and one control screen sample was not infected. Samples were harvested using trypsin gSYNC DNA extraction kit (Geneaid) PCR with Nextera overhangs