title: Dengue virus mutant
organism: Homo sapiens
description: Protocols: A pool of CRISPR mutagenized cells resisting a dengue virus mutant cytolytic infection was harvested from a live-dead genetic screen. 2 million 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.