CZ032_HIC
Crosslinking & Restriction Digest: Intact cells or nuclei are first fixed with formaldehyde to covalently crosslink spatially proximal chromatin regions. The crosslinked DNA is then digested with a restriction enzyme (e.g., MboI or DpnII with a 4-base pair recognition site) to create fragments with sticky ends.End Repair & Biotinylation: The digested ends are filled in with nucleotides, including a biotinylated residue (e.g., biotin-14-dCTP). This step marks the sites of cleavage and future ligation junctions.Proximity Ligation: Under highly diluted conditions that favor intramolecular ligation, the sticky ends of crosslinked fragments are joined. This key step creates chimeric DNA molecules from sequences that were physically close in the nucleus, regardless of their genomic linear distance.Reverse Crosslinking & DNA Purification: The protein-DNA crosslinks are reversed, and the DNA is purified. The biotin label is now specifically attached to the ligation junctions.Biotin Capture & Shearing: The purified DNA is sheared by sonication to a size appropriate for Illumina sequencing (~300-500 bp). Biotin-labeled fragments, which represent valid ligation products, are then selectively captured using streptavidin-coated magnetic beads.Illumina Library Construction: Standard Illumina library preparation steps are performed on-bead.