title: Genome-wide CRISPR screen for AAV2 infection in human cells
design: gDNA was isolated using the Qiagen DNA Blood Maxi kit (for reference cells) or multiple QIAamp DNA Mini kit columns (for sorted cells). CRISPR gRNA encoding DNA sequences were amplified in a two-step nested PCR using KAPA HiFi HotStart ReadyMixPCR Kit (Kapa Biosystems). In the first PCR step, 36-48 reactions and 12 reactions containing 6 g gDNA were set up for the reference and sorted samples, respectively, and amplified for 16 cycles. Reactions for each sample were pooled and mixed. In the second PCR step, 4 reactions containing 5 l PCR1 product were amplified for 12 cycles using indexed primers. PCR products were gel purified using QIAquick Gel Extraction Kit and sequenced on an Illumina NextSeq 500 using custom sequencing primers.