title: GSM3330957: MKN7 4C-seq; Homo sapiens; OTHER
library_construction_protocol: 10 million cells were cross-linked for 10 min with 1 % paraformaldehyde at RT, quenched with glycine, and lysed in 50 mL lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.5 % NP-40, 1 % TX-100 and 1X protease inhibitors) for 30 min. Nuclei were then digested by NLAIII enzyme followed by inactivation of the restriction enzyme by incubating at 65 °C for 20 min. The digested chromatin was subsequently ligated (circularized) overnight at 16 °C with 3000 U T4 ligase (NEB). Ligated chromatin was then reverse cross-linked by incubating with proteinase K at 65 °C and the RNA was removed by additional incubation at 37 °C with RNase A. The purified DNA was further digested with CSP6I restriction enzyme followed by circularization of the DNA. 4C products were subsequently amplified with bait-specific inverse primers, pooled and purified. Amplified bait-containing DNA fragments were ligated to NEB adaptors. Adaptor-ligated DNA was purified by Agencourt AMPure XP purification system (Beckman Coulter, A63881), PCR amplified (eight cycles) using NEB index primers, and sequenced on the Illumina HiSeq3000 to obtain 150 bp long reads.