GSM7989481: Input_screen2_01; Mus musculus; OTHER
Genomic DNA was extracted from cells using the GeneJET genomic DNA extraction kit (#K0721 Thermofisher) and the sgRNA library was amplified by a single-step PCR protocol (Broad Institute) for NGS using barcoded primers with staggered sequences. PCR product from multiple PCR reactions was pooled and gel purified and quantified by Qubit Fluorometer and subjected to sequencing on Illumina Nextseq 500 with 75 reads. Mouse Brie CRISPR knockout pooled library developed by David Root and John Doench was obtained from Addgene (Addgene #73633) was amplified in Stable 3 competent E. coli (Thermofisher) and subjected to next-generation-sequencing (NGS) to determine the distribution of sgRNAs in the library. To produce Lentivirus, 1.8 x 106 NIH 293T cells were seeded in six 10-cm plates and transfected the following day with 6 μg sgRNA pooled library in lentiGuide-Puro (Addgene# 73633), 6 μg psPAX2 plasmid, 1 μg pVSVG plasmid, and 0.024 mg polyethyleneimine (PEI). After 48 h, the lentivirus was harvested, centrifuged, and aliquoted into multiple 15 mL tubes and stored at -80o C or - 150o C. The functional titer of the lentivirus was determined by live-cell counting after puromycin selection. The parameters for how the library sequenced - chip: 75 high fidelity read