title: GSM6207566: ExtendFig3_Untreated-rep2_ND6-TAS.DL-30; Homo sapiens; OTHER
library_construction_protocol: 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 0.1% to 0.005%10. 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, M0515L) for approximately 10 cycles. Q5U is capable of reading and amplifying templates containing uracil bases and hence ensures accurate measurement of off-target editing level. 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 or MGISEQ-2000.