Amplicon Seq of 293T: Treatment condition 19
Genomic DNA from 293T cells was extracted 4 days post transfection and used for primary amplification of the mtORI and COX3 sites. Primary amplification using Phusion HS Polymerase Master Mix (Thermo Fisher Scientific F531S), 30 amplification cycles, 2ng of input genomic DNA (analyzed by Qubit; Themo Fisher Scientific Q33240), was conducted under manufacturer recommended conditions. The mtORI amplicon primers including i7/i5 adapters were GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCTCCTCGCTCCGGGCCCAT and TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGGGAACGTGTGGGCTATTTAGGCT. COX3 amplicon primers including i7/i5 adapters were GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCAATCACCTGAGCTCACCATAGTC and TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCGTGGAAGCCTGTGGCTA. The primary PCR products were purified using AmpureXP beads (Beckman Coulter A63880) with manufacturer recommended protocol (1.8x bead ratio) and measured for concentration using Qubit. Next, primary PCR products were diluted and samples of 2 ng of input DNA were indexed using i5/i7 Illumina indexing primers (Nextera XT Index Kit v2 Set A, FC-131-2001) over 12 cycles of amplification, using Phusion HF Polymerase Master Mix. Indexed libraries were then diluted and pooled according to Illumina recommended protocol for sequencing on Illumina NextSeq machine (300 cycles paired end). Amplicon sequencing data was analyzed using CRISPResso2 using the base editor output option, with paired end reads, --min_average_read_quality 28, and --min_single_bp_quality 13.