Long-read ChIA-PET for base-pair-resolution mapping of haplotype-specific chromatin interactions.
Document Type
Article
Publication Date
5-2017
JAX Source
Nat Protoc 2017 May; 12(5):899-915
Volume
12
Issue
5
First Page
899
Last Page
915
ISSN
1750-2799
PMID
28358394
Grant
Directors Innovation Fund, CA186714, 45DK107967, R25HG007631
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 andreads. Nat Protoc 2017 May; 12(5):899-915.
Recommended Citation
Li X,
Luo O,
Wang P,
Zheng M,
Wang D,
Piecuch E,
Zhu J,
Tian S,
Tang Z,
Li G,
Ruan Y.
Long-read ChIA-PET for base-pair-resolution mapping of haplotype-specific chromatin interactions. Nat Protoc 2017 May; 12(5):899-915