GSM5444406: A549-D30_Rep2; Homo sapiens; OTHER
Genomic DNA (gDNA) was extracted using the FastPure Cell/Tissue DNA Isolation Mini Kit (Vazyme, DC102) and DNA concentration was measured by Qubit using the Qubit™ dsDNA HS Assay Kit (Invitrogen). To generate sgRNA amplicons, we used a single-step PCR protocol which was adopted from the protocol published by Julia Joung et al 41. All the gDNA harvested from the screen was used for PCR amplification in 50 µl PCR reactions. Each reaction consisted of 2.5 µg gDNA plus water, 25 μL NEBNext Ultra II Q5 PCR Master Mix, 1.25 μL 10μM stagger forward primer, and 1.25 μL 10μM barcoded reverse primer. PCR reactions were cycling as follow: initial denature 3min at 98℃; followed by 30s denature at 98℃, 10s anneal at 63℃, 25s extension at 72℃, for 23 cycles; and final extension for 2min at 72℃. PCR products were size-selected using VAHTS DNA Clean Beads (Vazyme) according to the manufacturer's instructions and sequenced on a HiSeq2000 sequencer (Illumina).