![]() Haplotype-resolved chromatin interactions can be deduced if phased SNP information is available for the appropriate reference genome. The final tags (from all three categories of read pairs) are used to identify genomic binding sites of the protein of interest via peak calling. The final PETs are used to generate a 2D contact-map file for visualization and also to identify clusters of overlapping intrachromosomal loops. Each category is then aligned to a reference genome and only uniquely mapped and non-redundant tags are retained for further analysis. To analyze the data, reads pairs are first partitioned into three categories: (i) read pairs with no linker sequence, (ii) read pairs with a linker sequence and one usable genomic tag, or (iii) read pairs with a linker sequence and paired end tags (PETs). ![]() These fragments are subjected to PCR amplification with minimal cycles, size selection of the DNA fragments, and high-throughput paired-end sequencing. Streptavidin beads are then used to enrich for DNA fragments containing ligation junctions (i.e., containing the biotinylated bridge linker). The ligated DNA fragments are released by reverse cross-linking, and Tn5 transposase is used to simultaneously fragment the DNA and add sequencing adaptors. Next, pairs of DNA fragments are joined by proximity ligation with a “bridge linker” – a short double-stranded DNA sequence containing an internal biotinylated nucleotide and T overhangs on each end. Chromatin complexes immobilized on antibody beads are then subjected to DNA end repair and A-tailing. Immunoprecipitation (IP) is performed using a specific antibody to enrich for chromatin complexes involving a protein of interest. Then, nuclei are released by cell lysis and are sonicated to generate chromatin complexes containing DNA fragments. First, protein-DNA interactions are stabilized by dual-cross-linking in cells. ChIA-PET is a method for capturing genome-wide chromatin interactions that involve a protein of interest.
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