title: WGS of HAP1 TRIM28 KO
design: The cell lines were cultured following the providers (Horizon Discovery) recommendations, in IMDM medium supplemented with 10% FBS and 1% Penicillin/Streptomycin. Culture was maintained at 37C with 5% CO2 in a humidified atmosphere. Cells were passaged every 2-3 days. Cells were harvested at 70-80% confluence with 0.05% trypsin at 37C for 5 min. Dissociation of cells was checked using microscopy. Cells were split into two samples containing approximately 2x106 cells each, one of which was used for FACS analysis and the other for DNA extraction. Cells collected for FACS analysis were pelleted at 1000 rpm at 4C for 5 min, washed once in 500 ml of ice-cold PBS and resuspended in 250 L of ice-cold PBS. Cells were fixed in 750 L of ice-cold (-20C) ethanol with constant gentle vortexing and stored at 4C. Fixed cells were washed with 400 L of ice-cold PBS and centrifuged at 1000 rpm at 4C for 5 min. Cells were resuspended in 400 L of PBS, 400 L of Accutase and incubated at room temperature for 20 min. Cells were then pelleted and resuspended in 400 L of PBS. RNase A (10 mg/ml) treatment was done at 37C for 30 min. Propidium iodide at a concentration of 1 mg/mL was added and staining was done at room temperature in the dark for 30 min. Cells were passed through a polystyrene cell-strainer-capped tube before flow cytometry analysis. Analysis of PI-stained cells was done with a FACSAria Fusion Sorter. Cells of known ploidy (haploid/diploid) were used as controls for each run. DNA extraction was performed using the MasterPure TM Complete DNA &amp; RNA purification Kit following the manufacturers instructions.