Abstract

With the advent of massively parallel sequencing, considerable work has gone into adapting chromosome conformation capture (3C) techniques to study chromosomal architecture at a genome-wide scale. We recently demonstrated that the inactive murine X chromosome adopts a bipartite structure using a novel 3C protocol, termed in situ DNase Hi-C. Like traditional Hi-C protocols, in situ DNase Hi-C requires that chromatin be chemically cross-linked, digested, end-repaired, and proximity-ligated with a biotinylated bridge adaptor. The resulting ligation products are optionally sheared, affinity-purified via streptavidin bead immobilization, and subjected to traditional next-generation library preparation for Illumina paired-end sequencing. Importantly, in situ DNase Hi-C obviates the dependence on a restriction enzyme to digest chromatin, instead relying on the endonuclease DNase I. Libraries generated by in situ DNase Hi-C have a higher effective resolution than traditional Hi-C libraries, which makes them valuable in cases in which high sequencing depth is allowed for, or when hybrid capture technologies are expected to be used. The protocol described here, which involves 4 d of bench work, is optimized for the study of mammalian cells, but it can be broadly applicable to any cell or tissue of interest, given experimental parameter optimization.

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Acknowledgements

We thank S. Kasinathan and members of the Shendure laboratory for helpful comments on the manuscript. This work was funded by a National Institutes of Health (NIH) Director's Pioneer Award (1DP1HG007811Q14 to J.S.) and an NIH National Human Genome Research Institute (NHGRI) Genome Training Grant (5T32HG000035 to V.R.).

Author information

Affiliations

  1. Department of Genome Sciences, University of Washington, Seattle, Washington, USA.

    • Vijay Ramani
    • , Darren A Cusanovich
    • , Ronald J Hause
    • , Wenxiu Ma
    • , Ruolan Qiu
    • , William S Noble
    •  & Jay Shendure
  2. Department of Pathology, University of Washington, Seattle, Washington, USA.

    • Xinxian Deng
    •  & Christine M Disteche
  3. Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington, USA.

    • C Anthony Blau
    •  & Zhijun Duan
  4. Division of Hematology, University of Washington, Seattle, Washington, USA.

    • C Anthony Blau
    •  & Zhijun Duan
  5. Department of Medicine, University of Washington, Seattle, Washington, USA.

    • Christine M Disteche
  6. Howard Hughes Medical Institute, Seattle, Washington, USA.

    • Jay Shendure

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Contributions

V.R. and Z.D. developed the protocol. V.R., D.A.C., R.J.H., R.Q., and Z.D. performed experiments and optimized the protocol. W.M. and W.S.N. devised the processing pipeline for in situ DNase Hi-C data. X.D., C.A.B., C.M.D., W.S.N., J.S., and Z.D. supervised the research. V.R., J.S., and Z.D. wrote the manuscript, with input from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Jay Shendure or Zhijun Duan.

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    Supplementary Figure 1

    Relative abundance of ligation types in three biological replicate GM12878 libraries versus a Patski library

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https://doi.org/10.1038/nprot.2016.126

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