THP_PVL. THP_PVL

We would like to perform a genome-scale CRISPR knockout library screen to identify loss-of-function mutations conferring PVL resistance in a human macrophage model. The human genome-wide lentiviral gRNA library will be transduced into a Cas9-expressing THP-1 cells. Two independent infections will be conducted, thus producing two independent THP-1 mutant libraries. Following each transduction, cells will be drug-selected for 5-7 days. The surviving mutant cells will be split into two mutant pools to differentiate into macrophages using PMA. Mutant cells will be taken for genome DNA extraction, PCR and submitted for Illumina sequencing to determine the gRNA coverage before macrophage differentiation. After macrophage differentiation, the mutant cells will also be sequenced to determine the gRNA coverage after PMA treatment. Mutant macrophages will be treated with a lethal dose of PVL for 24 h. After 24 h, the cells will be stained with Annexin V/PI and sorted using a flow cytometer. The sorted cells will be lysed for genomic DNA, PCR and sequenced to identify/quantify the gRNAs in the PVL resistant cells. We aim to sequence the purified libraries on Illumina HiSeq 2500 by 50-bp single-end sequencing. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute please see http://www.sanger.ac.uk/datasharing/