Long-read ChIA-PET for base-pair resolution mapping of haplotype-specific chromatin interactions

Summary

Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a robust method to capture genome-wide chromatin interactions. Unlike other 3C-based methods, it includes a chromatin immunoprecipitation (ChIP) step that enriches for interactions mediated by specific target proteins. This unique feature allows ChIA-PET to provide the functional specificity and higher resolution for detecting chromatin interactions, whereas 3C/Hi-C approaches could not achieve. The original ChIA-PET protocol generates short paired-end tags (2×20 bp) to detect two genomic loci that are far apart on linear chromosomes but are in spatial proximity in the folded genome. We have improved the original approach by developing long-read ChIA-PET, in which the length of the paired-end-tags is increased (up to 2×250 bp). The longer PET reads not only improve the tag mapping efficiency but also increase the probability of covering phased SNPs, which allows haplotype-specific chromatin interactions identification. Here, we provide the detailed protocol for long-read ChIA-PET that includes cell fixation and lysis, chromatin fragmentation by sonication, ChIP, proximity ligation with bridge linker, Tn5 tagmentation, PCR amplification, and high-throughput sequencing. To a well-trained molecular biologist, it typically takes six days from cell harvesting to the completion of library construction, up to a further 36 hours for DNA sequencing, and less than 20 hours for processing raw sequencing reads.