title: Isolation of nuclei for use in genome-wide DNase hypersensitivity assays to probe chromatin structure.
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.
authors: Department of Biology, Boston University, Boston, MA, USA.
authors: Guoyu
authors: G
authors: Ling
authors: David J
authors: DJ
authors: Waxman
mesh_terms: D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002467:Cell Nucleus / Q000737:chemistry* / Q000235:genetics; D002843:Chromatin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D003850:Deoxyribonuclease I / Q000737:chemistry*; D004210:Dissection; D016678:Genome; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008099:Liver / Q000737:chemistry; D051379:Mice; D009690:Nucleic Acid Conformation; D017422:Sequence Analysis, DNA
chemical_list: D002843:Chromatin; D003850:Deoxyribonuclease I