title: GSM7324096: TIGER_HAP1_D0_R4; 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 960µg [Offtarget screen (1)] or 880µg [Tiger screens in HEK cells (2) and HAP1 cells (3)] gDNA for each sample. Per sample we performed 96 or 88 100µL PCR1 reactions with a 100µl reaction volume: 10µl 10x Taq buffer, 0.02U/µl Taq-B enzyme (Enzymatics #P7250L), 0.2mM dNTPs, 0.2µM forward and reverse primers and 100ng gDNA/µl; PCR1 thermocycling conditions were: 94°C/30s, 10x (screen 1) or 18x (screens 2 and 3) [94°C/10s, 55°C/30s, 68°C/45s], 68°C/3min. For each sample, all PCR1 products were pooled and thoroughly mixed before PCR2. For each sample, we performed 24 (screen 1) or 6 (screen 2 and 3) PCR2 reactions with a 100µl reaction volume: 20µl 5x Q5 buffer (NEB), 0.01U/µl Q5 enzyme, 20µl PCR1-product, 0.2mM dNTPs, 0.4µM forward and reverse PCR2-primers in 100µl. PCR2 thermocycling conditions were: 98°C/30s, 18x (screen1) or 8x (screen 2 and 3) [98°C/10s, 63°C/30s, 72°C/45s], 72°C/5min. For each sample, PCR2 products were pooled, followed by normalization (gel-based band densitometry quantification), before combining equal amounts of uniquely-barcoded samples. The pooled product was then purified using SPRI beads. First, we performed a 0.6x vol/vol SPRI to remove gDNA carryover, followed by addition of a 0.3x vol/vol SPRI (0.6 + 0.3 = 0.9x final) to the supernatant to purify the ~260bp PCR product. The final amplicons were sequenced on Illumina NextSeq 500 – II MidOutput 1x150 v2.5 (screen 1) and Illumina NextSeq 500 – II HighOutput 1x150 v2.5 (screen 2 and 3).