Sample preparation:
1. Follow the provided protocol or your original one to prepare homogenised cell samples from animal organs (e.g. bone marrow) or tissue culture treated with aldehydes (e.g., 0.6 mM formaldehyde for 1 h in our paper).
2. Lyse the cells with SDS buffer (2% SDS; 20 mM Tris, pH 7.5) and store them at -80°C.
DPC isolation and NGS library preparation:
1. Sonicate the sample using a sonicator (Covaris: 5% duty factor, 200 cycles per burst, 16 min at 25°C).
2. Add equal volume of KCl buffer (200 mM KCl; 20 mM Tris, pH 7.5).
3. Centrifuge the sample for 5 min at 20,000 g at 4°C.
4. Remove the supernatant and resuspend the pellet in 750 μL KCl buffer.
5. Incubate the sample at 55℃ for 5 min, followed by on ice for 1 min.
6. Centrifuge the sample for 5 min at 20,000 g at 4°C.
7. Repeat steps (4-6) 4 times.
8. Remove the supernatant and resuspend the pellet in 500 μL KCl buffer with 0.04 mg/ml Proteinase K.
9. Incubate the sample with gentle shake at 500 rpm for 3 h at 55°C, followed by on ice for 1 min.
10. Centrifuge the sample for 5 min at 20,000 g at 4°C and collect the supernatant to purify DNA using a QIAGEN PCR purification column.
11. Next-generation sequencing libraries are prepared using NEB Next Ultra II DNA Library Prep Kit for Illumina or MGIEasy Universal DNA Library Prep Set, according to the manufacturer’s instructions.