GSM7874167: hESC cells, TUBBP2 KO, Hi-C rep1; Homo sapiens; Hi-C
1) Lyse cells by grinding and lysing in an ice bath of lysate to obtain nuclei samples; 2) Restriction enzyme (DpnII) digestion was followed to break the original genomic DNA structure to form a molecular group in units of "protein-DNA cross-linking complex"; 3) Cleave the cleavage site using dNTP-labeled repair enzymes with biotin labeling; 4) Using DNA ligase treatment, the flat ends of DNA molecules on the same "protein-DNA crosslinking complex" were connected to each other, so that the DNA fragment reconstructs the structure based on spatial location to form Hi-C molecules. The junction of the reconstituted molecule would be marked with biotin; 5) Protease 65°C high temperature treatment, unlink protein cross-linking and release DNA, and then use phenolic chloroform extraction to obtain Hi-C characteristic DNA molecules; 6) Remove unsuccessfully ligated DNA fragment ends using T4 DNA polymerase exonuclease activity to eliminate residual biotin labeling. 7) Fragmented DNA molecules were interrupted using ultrasound, followed by terminal repair, A-tail ligation, streptavidin-activated magnetic beads to retrieve biotin marker molecules, sequencing linker ligation, PCR enrichment for library preparation, and finally recovered the fragments of interest using AMPure XP beads or agarose gel electrophoresis to obtain the final Hi-C library.