GSM6209755: Kidney cancer tissue replicate 1; Homo sapiens; OTHER
Cells were cross-linked with 0.5 % formaldehyde (Sigma) for 10 mins. The cross-linking reaction was terminated by glycine at a final concentration of 200 mM and lysed in lysis buffer (PBS contain 0.2% SDS) at room temperature for 5 min. After incubation, the nuclei were pelleted by centrifugation at 2,000 g/min for 5 min. The nuclei were transferred to 1.5 ml tubes and washed twice with PBS. For in situ restriction enzyme digestion, 140 μl of ddH2O, 20 μl of 10% Triton X-100, 20 μl of 10× NEBuffer 2.1, and 20 μl of HindIII (NEB, 20 units/μl) was added to the nuclei pellet and digested for 1.5 h at 37 °C in thermomixer (Eppendorf) with rotation at 1000 r.p.m. Add 5 μl of dNTP mix (10 mM each) and 5 μl of DNA polymerase I Klenow fragment (NEB, M0210) to the reaction system, place the sample in thermomixer with rotation at 37 °C at 1000 r.p.m for 30 mins. Add 27.5 μl of H2O, 3 μl of ATP (adenosine-triphosphate, 10mM), and 10 μl of T4 DNA ligase (Thermo, EL0011) to the reaction system, and placed the tube on the rotating mixers for 2 h at room temperature with rotation at 20 r.p.m. Add 20 μl of proteinase K (20 μg/ml) to the proximity ligation system, and then incubate at 60 °C for 2 hours. After digestion, the DNA was directly extracted using PCR Purification Kits (Zymo, D4013). DNA sequencing libraries were prepared using the VAHTS Universal Plus DNA Library Prep Kit (NDM627) according to manufacturer's protocol.