GSE176089
Detect-seq on transfected HEK293T cells [Targeted deep sequencing data]
We used a new technology, named Detect-seq, to perform genome sequencing on transfected HEK293T cells to see DdCBEs' off-target mutations. Besides, targeted-amplicon sequencing and ATAC-seq data were applied to validate the results of Detect-seq. Overall design: We applied DdCBEs to generate on-target editings on mt-DNA to see DdCBEs' editing performance and evaluate their effects on the whole mitochondrial genome. This is the Targeted deep sequencing(Target-seq) part. Target-seq was performed as previously reported. Primers containing the paired Illumina adaptor sequences in the overhangs were designed based on regions flanking the targeting sites (Supplementary table 2). A 10-nt barcode was also added into each primer pairs to reduce the detection limit from 10-1 to 10-5[nm in press]. 10-100 ng of genomic DNA was used for the first round of PCR amplification using NEBNext® Q5U® Hot Start HiFi PCR Master Mix (NEB, M0543L) for approximately 10 cycles. The PCR products were purified with 1× Agencourt AMPure XP beads and eluted in nuclease-free water. The second round of amplification was performed on the purified DNA samples with different index primers for about 15 cycles. The PCR products were purified with 0.8× Agencourt AMPure XP beads and eluted in nuclease-free water. The libraries were quantified using Qubit dsDNA HS Assay kit (Invitrogen) and Qubit 2.0 Fluorometer (INVITROGEN), and then pooled together for high-throughput sequencing by Illumina HiSeq X Ten.