title: GSM7750042: sgRNA from cells with highest 1% of fluorescence; Homo sapiens; OTHER
library_construction_protocol: Genomic DNA (gDNA) was isolated  using the Blood and Cell Culture DNA Maxi Kit (Qiagen, catalog no. 13362) . To amplify sgRNA sequences for Next Generation Sequencing (NGS), four parallel 100 μL PCR reactions were initially run for each condition, and pooled. Each 100 uL PCR reaction contained 1 - 2 ug of gDNA, Ex Taq polymerase (Takara Bio, catalog no. RR01CM) and an appropriate PCR primer pair. Amplified DNA was then indexed and barcoded for NGS in another PCR reaction including 5 uL of DNA from the initial PCR, Ex Taq polymerase, pooled P5 and barcoded P7 primers. DNA was then pooled and purified for sequencing using AMPure XP beads (Agencourt). 300 uL of pooled PCR product was mixed with 150 uL beads and incubated for 5 minutes to remove gDNA. Beads bound to gDNA were pelleted on the DynaMag-2 Magnet (Invitrogen) and the supernatant was then mixed with 540 uL of fresh beads and incubated for 5 minutes to bind the PCR products. After discarding the supernatant, beads were washed twice with 1 mL 70 % ethanol and then dried for approximately 5 min. Bound DNA was eluted from the beads using 300 uL sterile, nuclease-free water. Human Brunello genome-wide CRISPR KO pooled library (Addgene #73178) CRISPR