Genome-wide CRISPR/Cas9 screen in HEC1B isogenic cell lines

This study was designed to conduct the genome-wide CRISPR/Cas9 screen in HEC1B cells and its ARID1B knockout derivative cells. We conducted the screen in each cell line with TkoV3 genome-wide CRISPR/cas9 library at a coverage of 250x. Cells were infected with TKOv3 genome-wide library (Addgene, #90294; PMID: 28655737) at a transduction efficiency of 30% aiming 500 times representation of each sgRNA. Cells were selected with puromycin for two days and 20x10^6 cells collected as a baseline. Remaining cells were kept in culture for 18 days post-infection and collected as the endpoint. Genomic DNA was isolated using QIAamp DNA Blood Maxi Kit following manufacturer’s instructions. sgRNA library was amplified from genomic DNA using Illumina-compatible index primers and sequenced by NovaSeq platform aiming for 300-500 times read coverage. The sequencing data was analyzed statistically using the MAGeCK software to identify dependenciesCells were infected with lentiviral library, which were selected by puromycin 24 hours later. 3 days after puromycin selection, half of the cells were collected as baseline. The remaining cells were maintained for an additional two weeks for prolonged selection before harvesting as endpoints. Genomic DNA was extracted for library preparation and sequencing using Illumina.