title: CRISPR/Cas9 knockout screen of Homo sapiens: A375 melanoma cells treated with Ganoderic Acid DM
design: Genomic DNA was extracted from frozen A375 cell pellets using the Blood and Cell Culture DNA Maxi Kit (Qiagen, 13323). sgRNA regions were PCR-amplified using Q5 Hot Start High-Fidelity 2x Master Mix (NEB, M0494L) with 1.5 ug gDNA input per reaction. Ten parallel 50 uL PCRs were performed per sample using staggered forward primers (1-10 bp) for sequencing diversity and barcoded reverse primers for sample demultiplexing (primer details in Table S1). PCR conditions: 98 degrees C for 3 min; 28 cycles of 98 degrees C for 10 s, 60 degrees C for 30 s, 72 degrees C for 25 s; final extension 72 degrees C for 5 min. Pooled PCR products were purified, resolved on a 2% agarose gel, and the target band (~275 bp) was gel-extracted (Qiagen, 28704). Libraries were sequenced on the HiSeq-SE150 platform (NovogeneAIT Genomics, Singapore).