Amplicon sequencing of HEK293T cells: CD19+EGFP+
CD19 EGFP HEK293T cells were treated with 50 l Cas9-EDVs. Seven days post-treatment, cells were sorted for EGFP+ (on-target) and EGFP- (bystander) cells using a SH800S cell sorter (Sony Biotechnology). Genomic DNA was extracted from sorted HEK293T cells using QuickExtract (Lucigen) according to the manufacturers instructions. PrimeStar GXL DNA polymerase (Takara Bio) was used to amplify the B2M Cas9-RNP target site. The resulting PCR products were cleaned up using magnetic SPRI beads (UC Berkeley DNA Sequencing Facility). The Innovative Genomics Institute Next-Generation Sequencing Core performed library preparation and sequencing using a MiSeq V2 Micro 2x150bp kit (Illumina). Reads were trimmed using the BBDuk trimmer (Minimum Quality = 20, Minimum Length = 20 bp) and merged using BBMerge (Geneious Prime, version 2022.0.1). Trimmed and merged reads were analyzed with CRISPResso2.