title: GSM9566789: HCT116 cells transduced with GeCKO-v2 knockout pooled library, noAZ, Day14; Homo sapiens; OTHER
library_construction_protocol: To extract gDNA from the CRISPR/Cas9 screen cells, every 100 million cells were resuspended in 12 ml of NK lysis buffer (50 mM Tris-HCl pH 8.0, 50 mM EDTA, 1% SDS) and mixed with 120 µl of 10 mg/ml Proteinase K (Mesgen, MG0324). Cell lysis proceeded overnight at 55oC. Then, incubation with 60 µl of 20 µg/ml RNase A (ThermoScientific, #EN0531) was maintained for 30 minutes at 37oC, followed by precipitation with 4 ml of 7.5 M ammonium acetate (Sigma, #A1542). After centrifugation at 4000 x g for 30 minutes, the supernatant was collected and mixed with 12 ml of isopropanol before proceeding to centrifugation at 4000 x g for 10 minutes. The final DNA pellet was washed twice with 70% ethanol, air dried, and resuspended in 0.2X TE buffer. To ensure complete dissolution of genomic DNA, the solution was incubated at 65oC for 1 hour, followed by overnight incubation at room temperature prior to PCR amplification. The GeCKOv2 Human CRISPR Knockout Pooled Library was processed according to the manufacturer's protocols