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.
Department of Genome Sciences, University of Washington, Seattle, Washington, USA.
Vijay
0000-0003-3345-5960
V
Ramani
Department of Genome Sciences, University of Washington, Seattle, Washington, USA.
Darren A
DA
Cusanovich
Department of Genome Sciences, University of Washington, Seattle, Washington, USA.
Ronald J
RJ
Hause
Department of Genome Sciences, University of Washington, Seattle, Washington, USA.
Wenxiu
W
Ma
Department of Genome Sciences, University of Washington, Seattle, Washington, USA.
Ruolan
R
Qiu
Department of Pathology, University of Washington, Seattle, Washington, USA.
Xinxian
X
Deng
Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington, USA.|Division of Hematology, University of Washington, Seattle, Washington, USA.
C Anthony
CA
Blau
Department of Pathology, University of Washington, Seattle, Washington, USA.|Department of Medicine, University of Washington, Seattle, Washington, USA.
Christine M
CM
Disteche
Department of Genome Sciences, University of Washington, Seattle, Washington, USA.
William S
WS
Noble
Department of Genome Sciences, University of Washington, Seattle, Washington, USA.|Howard Hughes Medical Institute, Seattle, Washington, USA.
Jay
J
Shendure
Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington, USA.|Division of Hematology, University of Washington, Seattle, Washington, USA.
Zhijun
0000-0002-8147-793X
Z
Duan
D019921:Biotinylation; D002874:Chromosome Mapping / Q000379:methods*; D003851:Deoxyribonucleases / Q000378:metabolism*; D005557:Formaldehyde / Q000378:metabolism; D015723:Gene Library; D006801:Humans
D005557:Formaldehyde; D003851:Deoxyribonucleases
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