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Cold Spring Harb Protoc. 2018 Aug 1;2018(8). doi: 10.1101/pdb.prot104620.

Chromatin Interaction Analysis Using Paired-End-Tag (ChIA-PET) Sequencing in Tadpole Tissues.

Author information

1
Function and Mechanism of Action of Thyroid Hormone Receptor Group, UMR 7221 CNRS and Muséum National d'Histoire Naturelle, Sorbonne Universités, Paris 75005, France.
2
The Jackson Laboratory of Genomic Medicine, Farmington, Connecticut 06030.
3
The Department of Genetics and Developmental Biology, University of Connecticut, Farmington, Connecticut 06030.
4
Function and Mechanism of Action of Thyroid Hormone Receptor Group, UMR 7221 CNRS and Muséum National d'Histoire Naturelle, Sorbonne Universités, Paris 75005, France; sachs@mnhn.fr.

Abstract

Proper gene expression involves communication between the regulatory elements and promoters of genes. Today, chromosome conformation capture (3C)-based methods efficiently probe chromosome folding in the nucleus and thus provide a molecular description of physical proximity through DNA looping between enhancer(s) and their target promoter(s). One such method, chromatin interaction analysis using paired-end-tag (ChIA-PET) sequencing is a powerful high-throughput method for detection of genome-wide chromatin interactions. Following enrichment of the chromatin complexes with a dedicated antibody, through a process of immunoprecipitation (IP), DNA fragments are end-joined with specifically designed DNA-linkers through proximity ligation. The DNA-linkers contain the binding site for the type II restriction enzyme MmeI, which cleaves 20 bp from each end of the ligated fragments, thus releasing a "paired end tag" (PET): [20 bp tag]-[linker]-[20 bp tag]. The PETs are then deep-sequenced and reads are mapped to the reference genome, revealing both binding sites, as well as remote chromatin interactions mediated by the protein factors of interest. The method detailed here focuses on ChIA-PET library construction and can be completed in 2 wk.

PMID:
29895565
DOI:
10.1101/pdb.prot104620

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