Sanger sequencing of genomic DNA

A panel of 86 LCLs from the HapMap3 project was purchased from the Coriell Institute for Medical Research, Camden, NJ, USA. Three cell lines were selected for each of the three genotypes of a SNP. Genomic DNA was extracted using a Quick-DNA Miniprep Plus Kit (Zymo Research, Irvine, CA, USA).

PCR primers were designed to amplify the flanking areas of the target editing sites on the corresponding genomic DNA. Primers are: NDE1_Forward, 5′- CAACCAGGTGGAATCGTCTT-3′; NDE1_Reverse, 5′- ACTCGAACGCACCTCTAGGA-3′; ATM_Forward, 5′-CCAGGACAGCTACAGCATCA-3′; ATM_Reverse, 5′-CTAAGCCCTTCCCTTCCAAC-3′; MDM4_Forward, 5′-GTGATGGGGGATAGGGAGTT-3′; MDM4_Reverse, 5′-GCATTTCATCCCTCCTTTGA-3′; H2AFV_Forward, 5′-AGGCATGAGAATGACGTGAA-3′; H2AFV_Reverse, 5′-CTTCAACCTGGGCAAAAGAG-3′. PCR amplicons were purified by agarose gel electrophoresis and gel extraction using a PureLink® Quick Gel Extraction Kit (Invitrogen, Carlsbad, CA, USA), followed by Sanger sequencing to confirm the genomic sequence of the editing sites.