Mapping 3D genome architecture through in situ DNase Hi-C

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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Figure 1: A schematic overview of in situ DNase Hi-C.
Figure 2: Nuclei remain intact during the in situ DNase Hi-C protocol.
Figure 3: Digestion QC throughout the in situ DNase Hi-C protocol.
Figure 4: In situ DNase Hi-C results for the mouse embryonic kidney Patski cell line.

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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.).

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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.

Corresponding authors

Correspondence to Jay Shendure or Zhijun Duan.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Relative Abundances of ligation types in 3 biological replicate GM12878 libraries, vs. a Patski library

In situ DNase Hi-C reads demonstrate some cell-type specificity for the relative breakdown in long-range intrachromosomal read-pairs with respect to interchromosomal and short-range read-pairs, but these differences are reproducible, as shown when comparing three biological replicate libraries derived from the immortalized lymphoblastoid GM12878 cell line.

Supplementary information

Supplementary Figure 1

Relative abundance of ligation types in three biological replicate GM12878 libraries versus a Patski library (PDF 234 kb)

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Ramani, V., Cusanovich, D., Hause, R. et al. Mapping 3D genome architecture through in situ DNase Hi-C. Nat Protoc 11, 2104–2121 (2016). https://doi.org/10.1038/nprot.2016.126

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