CRISPR/Cas9 knockout screen of Homo sapiens: A375 melanoma cells treated with Ganoderic Acid DM
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).