<scp>ISDH</scp>‐seq: a robust methodology for profiling and characterization of open chromatin

Profiling of open chromatin is essential for understanding regulatory mechanisms that govern DNA-templated events. However, methodology-dependent detection of open chromatin sites (OCSs) has been reported in both humans and plants. Therefore, there is a pressing need for any reliable orthogonal methodologies to broaden the identification of open chromatin across the genome, particularly in plants. We here report the development of an in situ DNase I hypersensitivity sequencing (ISDH-seq) for the efficient characterization of open chromatin, which can be applicable to 50-200 K nuclei. This technique identifies 72% and 120% more OCSs than newly generated DNase- and ATAC-seq from the same batch of tissues as ISDH, respectively, in the rice genome. As compared to DH-specific OCSs, ISDH-specific OCSs exhibit distinct epigenetic features, including relative hypomethylation, a higher frequency of OCSs associated with H3K27me3 and increased associations with spatial chromatin interactions. Genes with H3K27me3-enriched ISDH-specific OCSs tend to be more expressed in a stress- and tissue-dependent manner, which have significant biological implications. The functions of ISDH-specific OCSs may be mediated by TF-centred networks or through chromatin loops. Importantly, a subset of genes co-regulated by more ISDH-specific distal OCSs (dOCSs) through chromatin loops are more evolutionarily conserved than genes regulated by a single ISDH-specific trans-OCS, including domesticated genes responsible for important agronomic traits. Thus, ISDH-seq can enrich and expand regulatory landscapes, facilitating a better understanding of their biological implications across plant genomes. It could serve as a reliable method that complements existing techniques and can be adapted for use in non-plant systems.