title: Genome-scale CRISPR knockout screen for Pteropine Orthoreovirus PRV3M
design: Genome-scale CRISPR knockout screen for Pteropine Orthoreovirus PRV3M
library_construction_protocol: A pool of mutagenized IGROV-1 cells were generated using the Brunello CRISPR knockout library, which was designed to target human genome with four unique sgRNA per gene. The mutagenized IGROV-1 cells were subjected to pteropine orthoreovirus PRV3M infection. After a total of two weeks of live/dead selection, mutagenized cells resisting cytolytic infection of pteropine orthoreovirus PRV3M was harvested fand subjected to NGS analysis. Resistant cells were harvested from each sub-library, and subjected to total genomic DNA extraction using DNA mini kit (Qiagen) according to the manufacturerвЂ™s instructions The sgRNA were amplified from gDNA in two-step PCR using Herculase II Fusion DNA Polymerase (Agilent). For first PCR, 20 ug of gDNA for each library was amplified for 16 cycles. For 2nd PCR, 1 reaction containing 5ul PCR product for each sub-library was amplified for 27 cycles, using Illumina indexed primers. PCR products were gel purified (Qiagen gel purification kit) and sequenced on Illumina MiSeq platform.