Nascent-hic-rep3 (CRX913074)
Cells were fixed with 1% formaldehyde and lysed in ice-cold Hi-C lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA630, 1x protease inhibitor cocktail) for 15 min. Pelleted nuclei were incubated in 0.5% sodium dodecyl sulfate at 62 degreeCC for 5 min. MboI restriction enzyme was added and chromatin was digested at 37 degreeCC for 5 hr. Biotin-14-dATP was used to mark the DNA ends followed by proximity ligation in intact nuclei. After crosslink reversal, DNA was sheared to a length of about 300 bp with Covaris M220. Biotin-labeled fragments were pulled down using Dynabeads MyOne Streptavidin C1 beads. The Hi-C libraries were amplified for 11-15 cycles with Q5 master mix following the operation manual. DNA was then purified and sequenced using NovaSeq platform.