GSM5930915: 4C-AG; Homo sapiens; OTHER
The 4C assay was carried out in SNP rs10486567 heterozygous cell clones, using a published protocol 61 with some modification. Briefly, 1 × 107 cells were cross-linked in 1 % formaldehyde at room temperature for 10 min and quenched by 0.125 M glycine. Nuclei were isolated in 5 ml of cold lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 5 mM MgCl2, 10 mM EGTA, 0.2% NP-40 and 1 mM PMSF) and then digested with 400 U HindIII(NEB, R3104S) at 37°C with rotation, followed by adding 4000 U T4 DNA ligase (NEB, M2020M) for in-situ ligation at 16°C overnight. The ligated DNA was de-crosslinked with proteinase K at 65 °C overnight. Purified DNA was digested with 100 U CviQ1 enzyme (NEB, RO639S) and then circularized using 4000 U T4 DNA ligase. Circularized DNA was amplified with SNP-specific inverse primers (see table s6). Each 4C library was sequenced as 150 bp paired-end reads using the Illumina Hiseq 4000 system.