GSM5808383: 4C DKO rep2; Homo sapiens; OTHER
40 million cells were cross-linked with 1% formaldehyde. The nuclei pellets were isolated by cold lysis buffer (10mM Tris-HCl, 10mM NaCl, 5mM EDTA, 0.5% NP 40) supplemented with protease inhibitors (Roche). The first digestion was performed overnight at 37°C with HindIII enzyme (NEB). Digestion efficiency was measured by gel electrophoresis. After confirmation of good digestion efficiency, DNA was ligated overnight at 16°C by T4 DNA ligase (Thermo Scientific) and de-crosslinked by proteinase k. After that, DNA was extracted by phenol-chloroform and this DNA was referred as “3C library”. The “3C library” was then processed for second digestion with DpnII enzyme (NEB) overnight at 37°C. After ligation, we obtained “4C template DNA. The concentration of the 4C DNA was determined by Qubit assays (Thermo Scientific). The 4C template DNA was then amplified using specific primers with Illumina Nextera adapters and sent for sequencing on the MiSeq platform.