GSM7235226: ARPE19 cells, Hi-C, normal control; Homo sapiens; Hi-C
2×107 cells were fixed in 1% formaldehyde for 10 min at room temperature and quenched with glycine. Fixed cells were permeabilized in 1 ml lysis buffer (10 mM Tris-HCl pH 8, 10 mM NaCl, 0.2% NP-40 and protease inhibitor cocktail (Sigma)) on ice for 15 min and spun down at 2,500 g for 5 min at 4℃. The pellet was resuspended in 345 μl of 1×NEBuffer r2.1 (NEB), and incubated with 38 μl of 1% SDS at 65℃ for 10 min. Next, 43 μl of 10% Triton X-100 was added to quench the SDS at 37℃ for 15 min. Then 12 μl of 10×NEBuffer r2.1 and 400 U HindIII (NEB) were added per aliquot, and mixed gently to digest the chromatin overnight at 37℃ on a rocking platform. On the next day, the HindIII restriction enzyme was inactivated at 65℃ for 25 min. Then, biotin-14-dATP (Life Technologies), dCTP, dGTP, dTTP and DNA polymerase I Klenow were added (NEB), and incubated at 37℃ for 2 h. The digested chromatin was diluted and re-ligated by T4 DNA ligase (NEB), incubated at 16℃ for 4 h, and shaken manually for three times. The chromatin products were de-cross-linked overnight by adding 50 μl of 10 mg ml-1 proteinase K (Sigma), and incubated at 65℃ overnight. DNA was extracted by phenol-chloroform extraction protocol. RNA was digested by adding 1 μl of RNase A (Sigma) and incubating at 37℃ for 30 min. Then T4 DNA polymerase (NEB) was used to remove biotin from un-ligation ends for 4 h at 20℃, and the enzymes were inactivated for 20 min at 75℃. DNA was purified and sheared to a size of 350 bp (Covaris M220). DNA size selection was conducted using AMPure XP beads (Beckman Coulter). Biotin-marked ligation products was isolated with MyOne Streptavidin C1 beads (Life Technologies). End repair, A adding, adaptor adding reaction, PCR amplification and DNA products size selection were performed sequentially. The libraries were sequenced by the Illumina PE150 paired-end sequencing platform.