Identification of active gene regulatory elements is a key to understanding transcriptional control governing biological processes such as cell-type specificity, differentiation, development, proliferation, and response to the environment. Mapping DNase I hypersensitive (HS) sites has historically been a valuable tool for identifying all different types of regulatory elements, including promoters, enhancers, silencers, insulators, and locus control regions. This method utilizes DNase I to selectively digest nucleosome-depleted DNA (presumably by transcription factors), whereas DNA regions tightly wrapped in nucleosome and higher-order structures are more resistant. The traditional low-throughput method for identifying DNase I HS sites uses Southern blots. Here, we describe the complete and improved protocol for DNase-seq, a high-throughput method that identifies DNase I HS sites across the whole genome by capturing DNase-digested fragments and sequencing them by high-throughput, next-generation sequencing. In a single experiment, DNase-seq can identify most active regulatory regions from potentially any cell type, from any species with a sequenced genome.
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