GSM8003945: Untreated 2; Mus musculus; OTHER
Total genomic DNA was isolated using NucleoSpin® Blood kits (Takara Bio Inc., USA; 740954 and 740950) and the concentrations of the isolated gDNA samples were measured using NanoDrop (Thermo Fisher Scientific, USA). The Mouse Brie CRISPR KO library carrying four gRNAs for each gene to cover the entire genome was a gift from David Root and John Doench (Addgene #73632 and #73633). The plasmid library was amplified and validated in the Center for Advanced Genome Engineering (CAGE) at St. Jude as described in the Broad GPP protocol, the only exception being the use of Endura DUOs electrocompetent E. coli cells. The St. Jude Hartwell Center Genome Sequencing Facility provided all NGS sequencing. Single end 100 cycle sequencing was performed on a NovaSeq 6000 (Illumina). Validation to check gRNA presence and representation was performed using calc_auc_v1.1.py (https://github.com/mhegde/) and count_spacers.py84. Viral particles were produced by the St. Jude Vector Development and Production laboratory using co-transfection of the lentiviral packaging plasmids along with the Brie library in the 293T cells. CRISPR KO screens were analyzed using Mageck-Vispr v0.5.7.