The purified ligation products are then digested by Tn5 transposase (tagmentation) for DNA library construction and high-throughput PET sequencing ( 3).īy design, the sequencing data derived from each ChIA-PET experiment contain three sets of genomic information ( 6): (i) the genome-wide binding profile by the protein factor of interest in the study, analogous to chromatin immunoprecipitation (ChIP)–PET ( 9) and ChIP sequencing (ChIP-seq) data ( 10) (ii) the chromatin interactions between the binding sites involving the protein factor and (iii) generic chromatin interactions that are not ChIP enriched, analogous to Hi-C data ( 11). Pairs of DNA fragments in each chromatin complex are joined via bridge linker ligation (proximity ligation). Immunoprecipitation is performed to enrich for chromatin complexes containing a specific protein of interest. First, chromatin interactions are stabilized via dual cross-linking by formaldehyde and ethylene glycol bis(succinimidyl succinate) (EGS), and then, the chromatin sample is fragmented by sonication. Briefly, long-read ChIA-PET is performed as follows. ChIA-PET has been applied to study long-range chromatin interactions in human and mouse cells and has provided key insights into the roles of a number of chromatin architecture proteins and transcriptional factors, including CCCTC-binding factor (CTCF) and RNA polymerase II (RNAPII), in human three-dimensional (3D) genome organization ( 4– 8).
Subsequently, an improved version for longer-read (2 × 150 bp) ChIA-PET was developed, which showed increased mapping accuracy and robustness ( 3). The first version of the ChIA-PET protocol extracted short paired tags for two contacting chromatin sites ( 2). ChIA-PIPE also supports the analysis of other related chromatin-mapping data types.ĬhIA-PET (chromatin interaction analysis with paired-end tags) enables genome-wide discovery of chromatin interactions involving a specific protein of interest ( 1). To enable structural interpretation, ChIA-PIPE calls chromatin contact domains, resolves allele-specific peaks and loops, and annotates enhancer-promoter loops. To enable visualization, ChIA-PIPE generates input files for two-dimensional contact map viewing with Juicebox and HiGlass and provides a new dockerized visualization tool for high-resolution, browser-based exploration of peaks and loops. ChIA-PIPE performs linker filtering, read mapping, peak calling, and loop calling and automates quality control assessment for each dataset. Here, we introduce ChIA-PIPE, a fully automated pipeline for ChIA-PET data processing, quality assessment, visualization, and analysis. Interpretation of ChIA-PET data requires a robust analytic pipeline. 3.ChIA-PET (chromatin interaction analysis with paired-end tags) enables genome-wide discovery of chromatin interactions involving specific protein factors, with base pair resolution.
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