Amplicon Sequencing of gRNAs from Brunello CRISPR mutagenized U87MG or eHAP cells after live/dead selection with Reovirus Type 3 Dearing Strain.

eHAP or U87MG cells were stably transduced with lentiCas9-Blast (Addgene, #52962) and subsequently selected using Blasticidin. Then, 300 million eHAP or U87MG cells that constitutively express Cas9 were transduced with lentiGuide-Puro from the Brunello library at MOI 0.3. Cells were then selected with puromycin, expanded to 3 billion cells, and then pooled together and cryofrozen in aliquots. One hundred million cells were thawed constituting over 1000× genome coverage worth of mutagenized library. Twenty T175 flasks were used for the U87MG-based screen, while twelve T175 flasks were used for the eHAP-based screen. The cells were allowed to recover for 48 hours before infecting with ReoT3D at an MOI of 0.1. Obvious CPE was observed within 72 hours. eHAP-resistant colonies were harvested two weeks later, while U87MG-resistant colonies were harvested six weeks later. The uninfected reference used was the unselected starting population. The unselected and selected cells were both processed with QIAamp DNA columns to purify the gDNA. 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. The amplicons were then subjected to next-generation sequencing on a HiSeq instrument lane (Illumina) via Novogene.