title: GSM8505615: JLat5A8 HuEpi AZD5582+IBET151 vRNA Rep1; Homo sapiens; OTHER
library_construction_protocol: Viral RNA was extracted using a QIAamp Viral RNA Mini Kit (Qiagen, Cat No 52904). Genomic DNA was extracted using a QIAamp DNA Blood Midi kit (Qiagen, Cat No 51183). Genomic DNA (gDNA) or reverse-transcribed viral RNA (vRNA) were amplified by a two-step PCR (PCR1: 12 cycles, PCR2: 20 cycles) to amplify 20bp sgRNA sequences in the HIV-CRISPR vector. All vRNA was amplified for each sample where appropriate. For gDNA samples, a total of 500-fold coverage of the 5,309 sgRNAs were amplified (10^6 cells were estimated to contain 6.6μg gDNA) with a maximum of 2μg gDNA per PCR reaction. For each sample, PCR1 reactions were pooled, cleaned up over a QIAquick PCR purification Kit (2 columns per sample), pooled again and used as template for PCR2. For vRNA, all PCR1 was amplified in the second round. For gDNA, 10 uL of pooled PCR1 was used as input for PCR2 (PCR2 primers include Illumina barcodes and adaptor sequences). Amplicons from PCR2 were pooled, purified by double-sided bead cleanup (AMPureXP, Beckman Coulter), quantified with a Qubit dsDNA HS assay and pooled for sequencing.