title: Long-read ChIA-PET for base-pair-resolution mapping of haplotype-specific chromatin interactions.
abstract: Chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) is a robust method for capturing 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 needed to detect chromatin interactions, which chromosome conformation capture (3C)/Hi-C approaches have not achieved. The original ChIA-PET protocol generates short paired-end tags (2 × 20 base pairs (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 single-nucleotide polymorphisms (SNPs), which allows haplotype-specific chromatin interactions to be identified. 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 a bridge linker, Tn5 tagmentation, PCR amplification and high-throughput sequencing. For a well-trained molecular biologist, it typically takes 6 d from cell harvesting to the completion of library construction, up to a further 36 h for DNA sequencing and <20 h for processing of raw sequencing reads.
authors.affiliation: The Jackson Laboratory for Genomic Medicine, Farmington, Connecticut, USA.|National Key Laboratory of Crop Genetic Improvement, College of Life Sciences &Technology, Huazhong Agricultural University, Wuhan, China.
authors.forename: Xingwang
authors.initials: X
authors.lastname: Li
authors.affiliation: The Jackson Laboratory for Genomic Medicine, Farmington, Connecticut, USA.
authors.forename: Oscar Junhong
authors.initials: OJ
authors.lastname: Luo
authors.affiliation: The Jackson Laboratory for Genomic Medicine, Farmington, Connecticut, USA.
authors.forename: Ping
authors.initials: P
authors.lastname: Wang
authors.affiliation: The Jackson Laboratory for Genomic Medicine, Farmington, Connecticut, USA.
authors.forename: Meizhen
authors.initials: M
authors.lastname: Zheng
authors.affiliation: The Jackson Laboratory for Genomic Medicine, Farmington, Connecticut, USA.
authors.forename: Danjuan
authors.initials: D
authors.lastname: Wang
authors.affiliation: The Jackson Laboratory for Genomic Medicine, Farmington, Connecticut, USA.|Department of Genetics and Genome Sciences, University of Connecticut Health Center, Farmington, Connecticut, USA.
authors.forename: Emaly
authors.initials: E
authors.lastname: Piecuch
authors.affiliation: The Jackson Laboratory for Genomic Medicine, Farmington, Connecticut, USA.|Department of Genetics and Genome Sciences, University of Connecticut Health Center, Farmington, Connecticut, USA.
authors.forename: Jacqueline Jufen
authors.initials: JJ
authors.lastname: Zhu
authors.affiliation: The Jackson Laboratory for Genomic Medicine, Farmington, Connecticut, USA.
authors.forename: Simon Zhongyuan
authors.initials: SZ
authors.lastname: Tian
authors.affiliation: The Jackson Laboratory for Genomic Medicine, Farmington, Connecticut, USA.
authors.forename: Zhonghui
authors.initials: Z
authors.lastname: Tang
authors.affiliation: National Key Laboratory of Crop Genetic Improvement, College of Life Sciences &Technology, Huazhong Agricultural University, Wuhan, China.
authors.forename: Guoliang
authors.initials: G
authors.lastname: Li
authors.affiliation: The Jackson Laboratory for Genomic Medicine, Farmington, Connecticut, USA.|National Key Laboratory of Crop Genetic Improvement, College of Life Sciences &Technology, Huazhong Agricultural University, Wuhan, China.|Department of Genetics and Genome Sciences, University of Connecticut Health Center, Farmington, Connecticut, USA.
authors.forename: Yijun
authors.initials: Y
authors.lastname: Ruan
mesh_terms: D000818:Animals; D002843:Chromatin / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D003584:Cytological Techniques / Q000379:methods*; D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D006239:Haplotypes; D006801:Humans; D011485:Protein Binding; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D002843:Chromatin; D004247:DNA