GSM8969091: S2 Mutant, Notch1 Promoter Viewpoint, rep2; Homo sapiens; OTHER
Ten million cells were crosslinked with 2% formaldehyde for 10 min, followed by a 5-min quenching with glycine. Cells were washed once with PBS and then incubated for 20 min in lysis buffer (10 mM Tris-HCl pH 8, 10 mM NaCl, 0.2% Igepal) with 1× protease inhibitors. First, three rounds of digestion were carried out with DpnII (NEB R0543S) at 37 °C in a thermomixer at 500 rpm (100 U for 4 h, 100 U overnight, and 100 U for another 4 h); the restriction enzyme was inactivated by heating to 62 °C for 20 min while shaking at 500 rpm. Next, the first ligation was performed with 2000 U of T4 DNA ligase (NEB M0202L) at 16 °C in 7 mL of Milli-Q water overnight. Samples were then phenol-chloroform extracted and ethanol precipitated. The second digestion was performed overnight with 50 U of NlaIII (NEB R0125S) or CviQI (NEB R0639S). The second ligation was carried out in a total volume of 5 mL with 3000 U of T4 DNA ligase (NEB M0202L). The ligated material was purified with SPRI beads and quantified using a Qubit dsDNA Assay Kit. The first round of PCR amplification with viewpoint-specific primers was performed using 4 × 50 µL PCR reactions with 200 ng of 4C template and 16 PCR cycles with Phusion polymerase (NEB M0530L). A second round of PCR with universal primers containing Illumina adapters was then performed, and the material was purified with SPRI beads. Two independent libraries per condition were sequenced on an Illumina HiSeq 4000 platform as single-end 150 bp reads.