Genome-wide CRISPR knockout screen to identify genes involved in macrophage differentiation
Genome-wide CRISPR knockout screen to identify genes involved in macrophage differentiation
The mouse Brie knockout CRISPR pooled library (Doench et al 2016) on the lentiGuide-Puro backbone was transduced into cas9-expressing conditionally-immortalized macrophage progenitors. Brie library transduced progenitors were differentiated into macrophages by removal of beta-estradiol and addition of MCSF. Cells were differentiated for seven days before harvest. Library samples were taken on day 0 of differentiation, and day 7 of differentiation. In addition, a sample of progenitors was not differentiated and passaged in parallel for 7 days. Three independent experiments were performed. Cells were lysed in 50mM Tris, 50mM EDTA, 1% SDS, pH 8 + 100ug/ml proteinase K, RNAse treated, then incubated with ammonium acetate for a final concentration of 1.875M. After centrifugation, supernatants were incubated with isopropanol to precipitate genomic DNA. After centrifugation gDNA pellets were washed in 70% ethanol. After a final centrifugation, gDNA pellets were air dried and resuspended in 10mM Tris HCL pH 8.5 + 0.1mM EDTA.  Library was constructed as previously described, using primers for the Brie library (Doench et al., 2016). Briefly the sgRNA loci were amplified by PCR using Illumina compatible primers that allow for sample multiplexing. Amplicons were purified then pooled. 