Rla_HiC
We collected tissue that was cross-linked for 10 minutes with 1% final concentration fresh formaldehyde and quenched with 0.2 M final concentration glycine for 5 minutes. The cross-linked cells were subsequently lysed in lysis buffer (10 mMTris-HCl (pH 8.0), 10 mM NaCl, 0.2% NP40, and complete protease inhibitors [Roche]). The extracted nuclei were resuspended with 150 L 0.1% sodium dodecyl sulfate (SDS) and incubated at 65C for 10 minutes. Then SDS molecules were quenched by adding 120 L water and 30 L 10% Triton X-100 and incubated at 37C for 15 minutes. The DNA in the nuclei was digested by adding 30 L 10x New England Biolabs (NEB) buffer 3.1 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 100 g/mL bovine serum albumin (BSA), pH 7.9) and 150 U of DPN II and incubated at 37C overnight. On the next day, the DPN II enzyme was inactivated at 65C for 20 minutes. Next, the cohesive ends were filled in by adding 1 L 10 mM dTTP, 1 L 10 mM dCTP, 1 L 10 mM dGTP, 2 L 5 mM Biotin-14-dATP, 14 L water, and 4 L (40 U) Klenow and incubated at 37C for 2 hours. Subsequently, 663 L water,120 L 10x blunt-end ligation buffer (300 mM Tris-HCl, 100 mM MgCl2, 100 mM DTT, 1 mM ATP, pH 7.8), 100 L 10% Triton X-100, and 20 U T4 DNA ligase were added to start proximity ligation. The ligation reaction was heldat 16C for 4 hour. After ligation, the cross-linking was reversed with 200 g/mL proteinase K (Thermo) at 65C overnight. DNA purification was achieved using QIAamp DNA Mini Kits (Qiagen) according to the manufacturer's instructions. Purified DNA was sheared to a length of 400 bp.