GSM5733052: Jurkat - High-Low pool - repl 3; Homo sapiens; OTHER
To extract genomic DNAs, 0.5 million cells were incubated with 20 ml, 10 mg/ml proteinase K (SIGMA-ALDRICH INC, P8044) in 300 ml cell swelling buffer (10mM Tris-HCl, pH 8.0, 85mM KCl, 0.5% NP-40, and 10mM MgCl2) at 55 °C overnight. Genomic DNA was extracted using Phenol:Chloroform:Isoamyl Alcohol (Life Technologies #15593031) and ethanol precipitation. The gRNA inserts were amplified from genomic DNA from multiple PCR reactions, in which 400 ng gRNA DNA was used in one single PCR reaction. For example, if there were a total 12 mg genomic DNA in one pool, 30 PCR reactions were performed using Herculase II fusion DNA polymerase (AGILENT TECHNOLOGIES, #600679) for 3 cycles to amplify guide insert and add Illumina TruSeq sequencing adaptors. For each pool, the amplified guide inserts from multiple PCR reactions were pooled together and purified by AMPure XP beads (BECKMAN COULTER). A second PCR was performed to add TruSeq sequencing primers (Forward primer: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTctTGTGGAAAGGACGAAAC; Reverse Primer: CAAGCAGAAGACGGCATACGAGATAAGGCCACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTttttAACTTGCTATTTCTAGCTCTAAAAC) to the purified guide inserts for 16 cycles, with 400 ng/PCR reaction. The final guide libraries were sequenced using an Illumina NextSeq instrument in pair-end mode with 75 bp read length.