title: GSM6234574: CRISPR-KO-screen GeCKO V2 libB Rep3 3cyt-hi 72h; Homo sapiens; OTHER
library_construction_protocol: Genomic DNA from 60M cells per condition was purified with a Quick-gDNA™ MidiPrep kit (Zymo Research). sgRNA library were amplified from the genomic DNA using a two-step nested PCR method modified from a previous published protocol (PMID: 28417999). Amplicons from the first step of PCR were gel purified and subjected to a second round of PCR to add Illumina sequencing adaptors and TruSeq indexes. Sequencing library amplified from the second round of PCR were size-selected and purified with a magnetic bead-based SPRIselect reagent (Beckman Coulter), and subjected to HiSeq4000 Illumina NGS platform using a single read (SR75) method.