Amplicon next-generation seq of HEK293T
For targeted deep sequencing analysis of base editing efficiency, the genomic DNAs were amplified by two rounds of PCR using KOD -Plus- Ver.2 DNA polymerase (Toyobo). First-round PCR was set up with 2L of cell lysate, 0.3 M of forward and reverse primers, 1 U of KOD DNA Polymerase, 0.2 mM dNTPs, 1 mM MgSO4. PCR was performed as follows: pre-denaturing (94 for 2min), 30 cycles of amplification (94 for 15 s, 60 for 30 s and 68 for 30 s) and final extension (68 for 5 min). Illumina barcodes were installed through second-round PCR. A 50L reaction was set up with 3L of the first-round PCR mixture as the template, 0.3 M of forward and reverse barcoding primers, 1 U of KOD DNA Polymerase, 0.2 mM dNTPs, 1 mM MgSO4. Second-round PCR was conducted as follows: pre-denaturing (94 for 2min), 30 cycles of amplification (94 for 15 s, 60 for 30 s and 68 for 30 s) and final extension (68 for 5 min). Equal amounts of PCR products were pooled together, and the mixture of PCR products was purified with FastPure Gel DNA Extraction Mini Kit (Vazyme, DC301). The purified PCR product mixture was sequenced by 150-bp paired-end reads Illumina Novaseq X plus platform (LianChuan).