Integration site library of WT MINT in HEK293T cells
We used aTn5-based method to enrich the integration site DNA and construct the sequencing library. Genomic DNA was extracted using a Cell/Tissue DNA extraction Kit (Bioteke) according to the manufacturers instructions. Subsequently, 100 ng of genomic DNA was tagmented by a Tn5 transposase (TransGen Biotech) pre-loaded with adapters. Following the tagmentation, the DNA was purified with 1  VAHTS DNA Clean beads, and 8.3 L of the eluted DNA was used as the template for the first round of PCR. The first round of PCR was performed with the P5-1 primer (specific to the P5 adapter) and a gene-specific primer (GSP1, targeting the 3' UTR of the donor RNA) using 2 x KAPA HiFi Hotstart ReadyMix (Roche) under the following conditions: 98C for 5 min; 15 cycles of [98C for 30 s, 70C (decreasing by 1C per cycle) for 2 min, 72C for 30 s; followed by 10 cycles of 98C for 30 s, 55C for 1 min, 72C for 30 s; and a final extension at 72C for 5 min. The PCR products were purified with 1.2  VAHTS DNA Clean beads and eluted in 20 L of nuclease-free water. The eluate served as the template for the second round of PCR, which employed the nested P5-2 primer and a GSP2 primer (containing sequences hybridizing to the 3' UTR and the P7 adapter). The reaction conditions for the second round PCR were: 98C for 30 s; 15 cycles of 98C for 10 s, 58C for 30 s, 72C for 60 s.