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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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.).
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.
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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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