GSM6886543: DMSO_2; Homo sapiens; OTHER
At endpoint, at least 3x10^7 cells per replicate were collected for genomic DNA extraction to ensure over 400-fold coverage of library using ZymoResearch Quick-gDNA MidiPrep Plus Kit (Zymo Research, Cat#ZYM-D4075-25TST). sgRNA sequences were amplified in two rounds: first round by GeCKO-F1/R1 primers using Herculase II Fusion DNA polymerase (Agilent Technologies, Cat #600679); second round by NGS-Lib_Fwd/Rev 1-10 indexed using NEBNext® High-Fidelity 2X PCR Master Mix (Cat #M0544L). PCR products were purified (Qiagen gel purification kit, Cat #28706, and Qiagen PCR purification kit, Cat #28106), quantified by Qubit (Thermo Fisher Scientific), and profiled by Fragment Analyzer. Samples were sequenced in one run on Illumina NextSeq 500 Instrument (Illumina) according to the manufacturer instructions, yeilding 20 to 48 mio single-end 75 nucleotide reads per sample.