GSM5830904: SCS-rep1; Escherichia coli; OTHER
E. coli DNA was extracted using Ezup Column Bacteria Genomic DNA purification kit (Sangon Biotech, Shanghai, China) according to the manufacture's recommended procedure.Mitochondrial DNA of HepG2 cells were harvested and washed once in PBS buffer. Mitochondrial DNA was extracted using the high purity plasmid DNA kit (Tsingke biotech, Beijing, China) according to the manufacture's recommended procedure. The extracted DNA was eluted in 200 µL H2O and further enriched by 0.4× KAPA pure beads (Roche). Genomic DNA was fragmented to obtain 200 to 500 bp fragments by using a JY92-II N Ultrasonic Homogenizer (Scientz) with the following settings: 130 W peak incident power for 48 cycles (1 cycle = 5 s on and 5 s off). The mixture (50 μL) of fragmented genomic DNA (10 ng) and spike-in DNA were end repaired and dA-tailed using Hieff NGS® Fast-Pace End Repair/dA-Tailing module (YEASEN Biotechnology Co., Ltd., Shanghai, China) according to the manufacture recommended protocol. As for the adapter ligation, 60 μL of end-repaired DNA, 5 μL of custom adapter (15 μM), 5 μL of Fast-Pace DNA ligase, and 30 μL of Fast-Pace DNA ligation Enhancer (YEASEN Biotechnology, Shanghai, China) were incubated at 20°C for 12 h in a 100-μL solution. The DNA was purified by 0.8× KAPA Pure beads (Roche) to remove the excess adaptor. The purified DNA was then denatured by heating at 95oC for 10 min in 10% DMSO (v/v) solution and chilling on ice for 5 min. The ABE8e deamination reaction was conducted in a 10-µL reaction mixture containing 10 ng DNA, 50 mM Tris-HCl (pH 7.5), 10 mM DTT, and 2.8 µM ABE8e. After incubation at 37oC for 4 h, the reaction was stopped by heating to 95oC for 10 min. The resulting DNA was purified by 0.8× KAPA Pure beads (Roche) to remove the DTT and ABE8e. The purified DNA was subjected to PCR amplification. The PCR solution (25 µL) included 10 µL of purified DNA, 12.5 µL of TSINGKE Master Mix (Tsingke Biotech), 1 μL of pre-P7 primer (10 μM), 1 μL of pre-P5 primer (10 μM), and 0.5 μL of H2O. The amplification program started at 98°C for 30 s, followed by 4 cycles at 98°C for 10 s, 60°C for 30 s, and 72°C for 1 min. PCR products were sequentially purified using 0.8× KAPA Pure beads (Roche) followed by the second round PCR amplification. The PCR solution (50 µL) included 20 µL of purified DNA, 0.5 µL of Phusion Plus DNA polymerase (Thermo Fisher Scientific), 10 µL of 5× Phusion Plus buffer, 10 µL of 5× Phusion GC Enhance, 1 μL of P7 primer (15 μM), 1 μL of P5 primer (15 μM), and 7.5 μL of H2O. The amplification program started at 98°C for 30 s, followed by 13 cycles at 98°C for 30 s, 72°C for 45 s, and ended with 1 cycle at 72°C for 5 min. The PCR products were purified using 0.8× KAPA Pure beads (Roche) and agarose gel using agarose gel extraction kit (Zymo Research).