title: p53-mediated DNA looping at CRMP4 promoter in colorectal cancer
design: p53-mediated DNA looping at CRMP4 promoter in colorectal cancer
library_construction_protocol: 1x 107 cells were crosslinked with 1% formaldehyde at room temperature for 10 min, and the reaction was quenched with 125 mM glycine. Nuclei were isolated and subjected to 2.5 U/μL micrococcal nuclease (MNase, Cat. No. M0247S, NEB) digestion for 10 min at 37 oC, and quenched by 4 mM EGTA. Chromatin fragments end repair was performed using T4 DNA polymerase (Cat. No. M0203S, NEB) and T4 polynucleotide kinase (Cat. No. M0201S, NEB) in the presence of dNTPs and ATP. Reactions were incubated at 20 °C for 30 min, allowing fill-in of 5'overhangs, removal of 3' overhangs, and phosphorylation of 5' termini. End-repaired chromatin was subsequently subjected to in situ ligation using T4 DNA ligase (Cat. No. M0202S, NEB). Crosslinks were reversed by overnight incubation at 65 °C in the presence of 100 U proteinase K (Cat. No. P8107S, NEB), and DNA was purified by phenol-chloroform extraction and ethanol precipitation. Forward and reverse primers were designed within the PBR1-4 regions to amplify ligation products derived from chromatin looping events. Circularized DNA templates were subjected to PCR amplification, and amplicons corresponding to approximately 150 bp were selected for library construction using NEBNext Ultra II DNA Library Prep Kit for Illumina (Cat. No. E7645S, NEB).