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


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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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




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).

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