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ChIA-PET is a method for detecting the pairwise chromatin interactions mediated by a specific protein of interest, and Long-Read ChIA-PET is the second version of this assay (and is used for the ChIA-PET experiments in the 4DN data portal). The name “long read” is relative to the original ChIA-PET protocol (Fullwood et al., 2009) that used a Type IIS restriction enzyme (MmeI) to generate “short read” (2x20 bp) paired-end tags (PET) from the chromatin DNA templates by proximity ligation. The key modifications of Long-Read ChIA-PET from the original ChIA-PET method are 1) the use of Tn5 transposase to cut the proximity-ligated chromatin DNA fragments randomly, generating longer DNA templates for sequencing analysis by paired-end reads (2x150 bp). Therefore, most of the PET reads are up to 200 bp in length by 2x200 bp paired End reading model by ILLUMINA sequencing. A minor change in the protocol is to use one “bridge” linker instead of two “A-B” linkers, further simplifying the protocol.
The protocol involves dual-crosslinking the cells with formaldehyde and EGS to fix potential contacts between physically proximal regions. Subsequently, both the cell and nuclear membranes are lysed, and the fragmentation step follows. An antibody is used to pull down interactions involving a specific protein factor. The resulting chromatin material goes through the proximity ligation by A-tailing and linker ligation. Reverse crosslinking removes the protein, leaving the DNA fragments to be amplified and sequenced. Finally, the processed data reveal protein binding intensity, pairwise loops, and genome-wide contact maps.
See Li et al., 2017 for more details on Long-Read ChIA-PET.