Abstract
DNase hypersensitivity (DHS) analysis coupled with high-throughput DNA sequencing (DNase-seq) has emerged as a powerful tool to analyze chromatin accessibility and identify regulatory sequences in genomic DNA on a global scale. In this method, intact nuclei are isolated from fresh tissue or cultured cells and then subjected to limited digestion using DNase I. The resulting short DNA fragments released by DNase digestion, which correspond to regions of open chromatin structure, are subsequently purified and identified by high throughput next generation DNA sequencing. This chapter describes methods used to isolate intact nuclei from mouse liver suitable for DNase-seq studies. The following chapter presents a detailed protocol for DNase I digestion of liver nuclei followed by the isolation of DNase-released fragments for sequencing and genome-wide mapping of DHS sites.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Bell O, Tiwari VK, Thomä NH, Schübeler D (2011) Determinants and dynamics of genome accessibility. Nat Rev Genet 12:554–564
Gross DS, Garrard WT (1988) Nuclease hypersensitive sites in chromatin. Annu Rev Biochem 57:159–197
Boyle AP, Davis S, Shulha HP, Meltzer P, Margulies EH, Weng Z, Furey TS, Crawford GE (2008) High-resolution mapping and characterization of open chromatin across the genome. Cell 132:311–322
Crawford GE, Davis S, Scacheri PC, Renaud G, Halawi MJ, Erdos MR, Green R, Meltzer PS, Wolfsberg TG, Collins FS (2006) DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays. Nat Methods 3:503–509
Sabo PJ, Kuehn MS, Thurman R, Johnson BE, Johnson EM, Cao H, Yu M, Rosenzweig E, Goldy J, Haydock A, Weaver M, Shafer A, Lee K, Neri F, Humbert R, Singer MA, Richmond TA, Dorschner MO, McArthur M, Hawrylycz M, Green RD, Navas PA, Noble WS, Stamatoyannopoulos JA (2006) Genome-scale mapping of DNase I sensitivity in vivo using tiling DNA microarrays. Nat Methods 3:511–518
Song L, Crawford GE (2010) DNase-seq: a high-resolution technique for mapping active gene regulatory elements across the genome from mammalian cells. Cold Spring Harb Protoc 2010:pdb.prot5384
Ling G, Sugathan A, Mazor T, Fraenkel E, Waxman DJ (2010) Unbiased, genome-wide in vivo mapping of transcriptional regulatory elements reveals sex differences in chromatin structure associated with sex-specific liver gene expression. Mol Cell Biol 30(23): 5531–5544
Lichtsteiner S, Wuarin J, Schibler U (1987) The interplay of DNA-binding proteins on the promoter of the mouse albumin gene. Cell 51:963–973
Acknowledgments
Supported in part by NIH grant DK33765 (to DJW).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Ling, G., Waxman, D.J. (2013). Isolation of Nuclei for Use in Genome-Wide DNase Hypersensitivity Assays to Probe Chromatin Structure. In: Bina, M. (eds) Gene Regulation. Methods in Molecular Biology, vol 977. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-284-1_2
Download citation
DOI: https://doi.org/10.1007/978-1-62703-284-1_2
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-283-4
Online ISBN: 978-1-62703-284-1
eBook Packages: Springer Protocols