GSM3901607: 1μMGSK343treatment_K562_DMSO_HOXD13_rep2_4Cseq; Homo sapiens; OTHER
Briefly, 4 × 107 cells were cross-linked with 1% formaldehyde. The nuclei pellets were isolated by cell lysis with cold lysis buffer (10mM Tris-HCl, 10mM NaCl, 5mM EDTA, 0.5% NP 40) supplemented with protease inhibitors (Roche). First step digestion was performed overnight at 37°C with HindIII enzyme (NEB). Digestion efficiency was measured by RT-qPCR with HindIII site–specific primers. After confirmation of good digestion efficiency, DNA was ligated overnight at 16°C by T4 DNA ligase (Thermo Scientific) and de-crosslinked. Following de-crosslinking, DNA was extracted by phenol-chloroform. The DNA was then processed for second digestion with DpnII enzyme (NEB) overnight at 37°C. After final ligation, 4C template DNA was obtained and the concentration was determined using 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 system.