Amplicon Sequencing of gRNAs from Brunello CRISPR mutagenized A549 and U87-MG cells after live/dead selection with EV-D68 IL
Amplicon Sequencing of gRNAs from Brunello CRISPR mutagenized A549 and U87-MG cells after live/dead selection with EV-D68 IL
>500-fold coverage of mutagenized cells (Brunello library) was infected with EV-D68 IL (MOI 0.1). Resistant cell colonies were harvested. Uninfected starting populations of mutagenized cells were used as the unselected reference.  Total genomic DNA from both virus-resistant and uninfected cells was respectively extracted using QIAamp DNA Mini Kit (Qiagen). A first round of PCR was used to amplify the guide RNA sequences encoded in the gDNA, followed by a second round of PCR to add the barcodes/adapters for amplicon sequencing. 2% agarose gels and a QIAquick gel extraction kit were used to purify the amplicons.