DNase I hypersensitive site (DHS) mapping combined with high-throughput sequencing (DNase-seq) enables the identification of cis-regulatory DNA elements (CREs) genome wide. However, despite the wide applications of DNase-seq in plants, its application to the highly repetitive genomes of plants has lagged. Here, we describe a modified DNase-seq method, making it more practical for application to plants with genomes enriched with repetitive DNA. This approach adopts a double-hit-based strategy, in which small (<250-bp) DNA fragments digested by DNase I are selected and used for sequencing library construction. Using these protocols, we have conducted DNase-seq in plants with high content of repetitive DNA, including maize, sugarcane, and tetraploid cotton. Genome-wide maps of DHS and CREs have been created using these DNase-seq datasets. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Nuclei isolation Basic Protocol 2: DNase I digestion Basic Protocol 3: Target DNA isolation Basic Protocol 4: Library construction and validation.
Keywords: DNase I hypersensitive site; DNase-seq; cis-regulatory DNA element; double-hit; open chromatin; plant; transcription factor.
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