title: Genome-wide CRISPR knockout screen in Mycobacterium tuberculosis infected macrophages to identify regulators of TNF and iNOS expression
library_construction_protocol: 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. Macrophages were infected with M. tuberculosis. 24 hours post infection cells were fixed and stained for expression of TNF and iNOS. A sample of unsorted cells was collected, and remaining cells were FACS sorted into two groups: TNF positive and iNOS negative, or TNF positive and iNOS positive. Two 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 centifugation 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. 