title: GSM7324069: Offtarget_HEK293FT_D15_R3; Homo sapiens; OTHER
library_construction_protocol: Genomic DNA was extracted from screen cells using the following protocol: Per 100 million cells, 12 mL of NK Lysis Buffer (50 mM Tris, 50 mM EDTA, 1% SDS, pH 8) was used for cell lysis. Once cells were resuspended, 60 µL of 20 mg/ml Proteinase K (Qiagen) was added and the sample was incubated at 55°C overnight. The next day, 60 µL of 20 mg/ mL RNase A (Qiagen) was added, mixed, and samples were incubated at 37 C for 30 min. Then 4 mL of pre-chilled 7.5 M ammonium acetate was added, samples were vortexed and spun at 4,000 g for 10 min. The supernatant was placed in a new tube, mixed well with 12 mL isopropanol and spun at 4,000 g for 10 min. DNA pellets were washed with 12 mL of 70% ethanol, spun, dried and pellets were resuspended with 0.2x TE buffer (Sigma). In addition, we also generated linearized plasmid library input and diluted it down to mimic similar copy number conditions as the genomic DNA samples. We used a two-step PCR protocol (PCR1 and PCR2) to amplify the guide RNA cassette for Illumina sequencing from genomic DNA (gDNA). For the PCR1 reaction, we used 960µ