title: genome-wide CRISPR screening of HIV-GFP Jurkat cells
design: The human CRISPR knockout pooled library (Brunello, two vector system) purchased from Addgene was used for the genome-wide CRISPR screens. The standard procedures in the screens was performed as described previously 47,48. The CRISPR sgRNA library virus was packaged by transfecting HEK293T cells with library DNA, pMD2.G and a packaging vector pC-Help. After latent cells described in Figure 1a was sorted , 2.4 x 108 of the cells were infected at a low multiplicity of infection (0.30.5) to ensure that most cells receive only 1 viral construct with high probability. Two days after transduction, cells were selected in medium containing 1 g ml1 puromycin for two weeks to obtain the pooled knockout cells of latently HIV-infected cells. Then, 5 x 107 pooled knockout modified Jurkat cells were stimulated with either 0.1 M SAHA or 5 nM AZD5582. GFP+ cells were sorted by FACS. The sorted cells and and the control cells (transduced with the sgRNA library, cultured in parallel but without infection/sorting) were immediately lysed for genomic DNA extraction. The PCR to amplify the sgRNA was performed by using primers suggested by Brunello library inventors. The PCR products were purified by gel extraction, and the samples were deep-sequenced by Illumina Nextseq. Each library was sequenced at 30~40 million reads to achieve ~300X average coverage over the CRISPR library.