GSM1908921: HiC-E14_0915; Mus musculus; OTHER
A new Hi-C pipeline was applied according to the recent development3. Briefly, 2 million cells were cross-linked with 1% formaldehyde for 10min at RT and reaction was quenched using 125 mM of Glycine for 5 min at RT. Nuclei were isolated and directly applied for digestion using 4 cutter restriction enzyme MboI (NEB) at 37 °C o/n. The single strand overhang was filled with biotinylated-14-ATP (Life Tech.) using Klenow DNA polymerase (NEB). Different from tradition Hi-C, with in situ protocol the ligation was performed when the nuclear membrane was still intact. DNA was ligated for 4h at 16 °C using T4 ligase (NEB). Protein was degraded by proteinase K (NEB) treatment at 55 °C for 30 min. The crosslinking was reversed with 500 mM of NaCl and heat at 68 °C o/n. DNA was purified and sonicated to 300-700 bp small fragments. Biotinylated DNA was selected with Dynabeads My One T1 Streptavidin beads (Life Tech.). Sequencing library was prepared on beads and intensive wash was performed between different reactions. TruSeq