Amplicon sequencing of human T cells: CAR+
Human peripheral blood mononuclear cell-engrafted NSG mice (745557, Jackson Laboratory) were injected with 100 l of Cas9-EDVs, lentivirus, or phosphate-buffered saline (PBS) by retro-orbital injection following anesthesia induction with 2-3% isofluorane. 10 days post-treatment, mice were euthanized with CO2 to harvest spleen. Single-cell suspensions of spleen were prepared and sorted for mCherry+ and mCherry- primary human T cells that were either expressing CD4 or CD8 using a SH800S cell sorter (Sony Biotechnology). Genomic DNA was extracted from sorted mCherry+ and mCherry- primary human T cells using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturers instructions. PrimeStar GXL DNA polymerase (Takara Bio) was used to amplify the TRAC 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 NextSeq 1000/2000 P2 V3 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.