GSM8125114: KIT_multiAsCas12a-gCH130; Homo sapiens; OTHER
For analysis at the CD55 and CD81 loci, genomic DNA was isolated using NucleoSpin Blood (Macherey-Nagel, Catalog no. 740951.50). For analysis at the KIT locus, cell lystates were generated using QuickExtract DNA Solution (Lucigen) and directly subjected to PCR amplification. For analysis of CD55 and CD81 loci, PCRs for loci of interest were run using Amplicon-EZ (Genewiz) partial Illumina adapters and amplicons were processed using NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Catalog no. 740609.250). Paired end (2 x 250 bp) sequencing was completed at GENEWIZ (Azenta Life Sciences). For analysis of the KIT locus, cell lysates were subjected to 15 cycles of PCR to introduce Illumina sequencing primer binding sites and 0-8 staggered bases to ensure library diversity. After reaction clean-up using ExoSAP-IT kit (Thermo Fisher 78201), an additional 15 cycles of PCR was used to introduce unique dual indices and Illumina P5 and P7 adaptors. Libraries were pooled and purified by SPRIselect magnetic beads before paired-end sequencing using an Illumina MiSeq. Sequencing primer binding sites, unique dual indices (from Illumina TruSeq kits), P5 and P7 adaptor sequences are from Illumina Adaptor Sequences Document # 1000000002694 v16. PCR amplicon sequencing