GSM8620442: K562,EC100-GFP;RAD21-AID,DMSO,rep2,GFP; Homo sapiens; OTHER
4C templates preparation was performed as described in PMID: 31351925. In short, 5-10M cells were crosslinked with 2% formaldehyde for 10 minutes and quenched with 0.125M final concentration glycine on ice. Pellets were resuspended in lysis buffer (50 mM Tris-HCl pH 7.5, 0.5% NP-40, 1% TritonX-100, 150 mM NaCl, 5 mM EDTA) for 20 minutes on ice. Then, SDS (0.3% final concentration) was added, followed by TritonX-100 (2.5% final concentration), and chromatin was digested in situ using 100U Csp6I (ThermoFisher Cat#ER0211). DNA was then ligated using T4 ligase (Roche Cat#10799009001), decrosslinked by overnight ProtK treatment (Roche Cat#3115801001), and purified using isopropanol precipitation. Samples were then digested using 50U NlaIII (NEB Cat#R0125), ligated at a concentration of 5ng template/ul, and final 4C templates were purified with P-Beads (Macherey Nagel Cat#744100.34). 4C PCRs were performed in a two-step manner using the Expand Long Template PCR system (Roche Cat#11759060001). Indexed Illumina sequencing adapters were introduced to ligation fragments of interest with a two-step PCR strategy using ELT PCR System (Roche, cat. no. 11681842001).