GSM9079253: 4C-seq ΔCTRL2 viewpoint 1 replicate 1; Homo sapiens; OTHER
Cells were crosslinked with 2% formaldehyde for 10 min and quenched with 0.125 M glycine. After washing with PBS, cell pellets were lysed in buffer with Tris-HCl, NaCl, NP-40, Triton X-100, EDTA, PMSF, and protease inhibitors. Nuclei were isolated for chromatin digestion and ligation. For 4C-seq, chromatin was first digested with HindIII, heat inactivated, and ligated with T4 DNA ligase overnight. Crosslinks were reversed, and a second digestion was performed using DpnII followed by a second ligation to generate circular DNA. Inverse PCR using bait-specific primers amplified 4C fragments. Barcodes were added in a second PCR round. For UMI-4C, chromatin was digested with DpnII, ligated, purified, and then sonicated to 450–550 bp. Illumina adapters were ligated, and bait-specific nested PCR was performed to generate 500 bp libraries. Dual-index barcodes were added before sequencing.