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in situ ChIA-PET
In situ ChIA-PET is an improved protocol with the same goal as its predecessor Long-Read ChIA-PET, but with higher efficiency. This method detects the pairwise chromatin interactions mediated by a specific protein of interest in vivo — directly in the cell nucleus. The protocol was developed specifically for the 4DN and ENCODE consortia in
Inspired by the in situ Hi-C method (Rao et al., 2014), the in situ ChIA-PET technique employs the nuclear permeabilization and restriction enzyme (AluI) digestion steps to keep the chromatin structures intact within the nucleus. The A-tailing and proximity ligation are performed inside the cell nucleus, followed by the immunoprecipitation step. Resulting materials are tagmented, amplified, and sequenced. Finally, the processed data reveal protein binding intensity, pairwise loops, and genome-wide contact maps. A major advantage of this method over the Long-Read ChIA-PET is its ability to efficiently capture the meaningful intra-chromosomal loops, owing to the in situ digestion and ligation techniques.