GSM8224974: K562 cells DUBR TSS mutant 2,  BBQ locus viewpoint, biol_rep 1; Homo sapiens; OTHER
10 million cells were crosslinked with 2% formaldehyde for 10 minutes, followed by 5-minute quenching with glycine. Cells were washed once with PBS, and then cells were incubated for 20 min in lysis buffer (10 mM Tris-HCl pH 8, 10 mM NaCl, 0.2% Igepal) with 1x protease inhibitors. For the first digestion, three rounds of digestion were made with DpnII (NEB R0543S) at 37 C in a thermomixer at 500 RPM (100U for 4 hours, 100U overnight, and 100U for another 4 hours); the restriction enzyme was inactivated by heating to 62 C for 20 minutes while shaking at 500 rpm. Then the first ligation was made with 2000U of T4 DNA ligase (NEB M0202L) at 16 C in 7mL of Milli-Q water overnight. Samples were then phenol-chloroform extracted and ethanol precipitated, and the second digestion was performed overnight with 50U of NlaIII (NEB R0125S) or HindIII (NEB R0104S). The second ligation was performed in 5mL total with 3000 units of T4 DNA ligase (NEB M0202L). Libraries were constructed starting from the second ligation material which was purified with SPRI beads, and it was quantified with a Qubit dsDNA Assay Kit. A first round of PCR amplification with viewpoint-specific primers  was performed with 4 x 50 µL PCR reactions with 200ng of 4C template using 16 PCR cycles and the Phusion polymerase (NEB M0530L). A second round of PCR with universal primers containing Illumina adapters was performed, and the material was purified with SPRI beads.