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Long-read ChIA-PET for base-pair-resolution mapping of haplotype-specific chromatin interactions

Nature Protocols volume 12, pages 899–915 (2017)Cite this article

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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.

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Figure 1: Schematic of long-read ChIA-PET protocol.
Figure 2: Flowchart of key steps and quality control (QC) in long-read ChIA-PET protocol.
Figure 3: Long-read ChIA-PET data-processing workflow and data features.

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Acknowledgements

Y.R. is supported by the Director Innovation Fund of The Jackson Laboratory; grant nos. NCI R01 CA186714, NHGRI R25HG007631, and NIDDK U54DK107967 (4DN); and the Roux family endowment. X.L. is supported in part by China's '111 project' (grant no. B07041). G.L. is supported by the National Natural Science Foundation of China (grant no. 91440114) and the Fundamental Research Funds for the Central Universities (grant no. 2662014PY001).

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Author notes

  1. Oscar Junhong Luo

    Present address: Present address: Transformational Bioinformatics, Health and Biosecurity Business Unit, Commonwealth Scientific and Industrial Research Organisation, North Ryde, New South Wales, Australia.,

  2. Xingwang Li and Oscar Junhong Luo: These authors contributed equally to this work.

Authors and Affiliations

  1. The Jackson Laboratory for Genomic Medicine, Farmington, Connecticut, USA

    Xingwang Li, Oscar Junhong Luo, Ping Wang, Meizhen Zheng, Danjuan Wang, Emaly Piecuch, Jacqueline Jufen Zhu, Simon Zhongyuan Tian, Zhonghui Tang & Yijun Ruan

  2. National Key Laboratory of Crop Genetic Improvement, College of Life Sciences & Technology, Huazhong Agricultural University, Wuhan, China

    Xingwang Li, Guoliang Li & Yijun Ruan

  3. Department of Genetics and Genome Sciences, University of Connecticut Health Center, Farmington, Connecticut, USA

    Emaly Piecuch, Jacqueline Jufen Zhu & Yijun Ruan

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  1. Xingwang Li
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Contributions

X.L. and Y.R. designed the experimental part of the protocol; O.J.L. and G.L. designed the data-processing pipeline and analyzed the data with assistance from S.Z.T. and Z.T.; X.L. implemented the protocol with assistance from P.W., M.Z., D.W., E.P. and J.J.Z.; X.L., O.J.L. and Y.R. wrote the manuscript with input from G.L.

Corresponding author

Correspondence to Yijun Ruan.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Software 1

Perl script 1: prepare_reads_single.pl. (TXT 5 kb)

Supplementary Software 2

Perl script 2: prepare_reads_pair.pl. (TXT 1 kb)

Supplementary Software 3

Perl script 3: clustering_bridge.pl. (TXT 2 kb)

Supplementary Software 4

Python script 1: fetch_base_snp_v081.py. (TXT 1 kb)

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Li, X., Luo, O., Wang, P. et al. Long-read ChIA-PET for base-pair-resolution mapping of haplotype-specific chromatin interactions. Nat Protoc 12, 899–915 (2017). https://doi.org/10.1038/nprot.2017.012

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