alias: GSM3473798
title: GSM3473798: THP_BRUVSVG_viralRNA_1; Human immunodeficiency virus; OTHER
library_construction_protocol: Viral RNA was extracted using a QIAamp Viral RNA Mini Kit (Qiagen) Genomic DNA or reverse-transcribed viral RNA were amplified by a two-step PCR (PCR1: 12 cycles, PCR2: 20 cycles) to amplify 20bp sgRNA sequences in the HIV-CRISPR vector. All Viral RNA was amplified for each sample where appropriate. For Genomic DNA samples a total of 500-fold coverage of the 15,414 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 viral RNA, all PCR1 was amplified in the second round. For gDNA, 5uL 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 SPRI (AMPureXP, Beckman Coulter), quantified with a Qubit HS dsDNA assay and pooled for sequencing.
identifiers.namespace: GEO
identifiers.id: GSM3473798