ISDH-seq: a robust methodology for profiling and characterization of open chromatin

We present in situ DNase I hypersensitivity sequencing (ISDH-seq), a novel methodology for genome-wide open chromatin characterization in plants, which can be applicable to 100-200K nuclei. Compared to conventional DNase-seq and ATAC-seq applied to the same rice tissues, ISDH-seq detects 72% and 120% more open chromatin sites (OCSs), respectively. ISDH-specific OCSs exhibit unique epigenetic signatures: hypomethylation, co-enrichment with H3K27me3, and strong spatial chromatin interaction associations.ISDH-seq overcomes method-specific biases in OCS detection, expands the plant regulatory genome atlas, and offers cross-species applicability. This technology provides critical insights into noncoding regulatory mechanisms and their biological implications in plant genomes. Overall design: This study profiles open chromatin landscapes in Oryza sativa (rice) using two-week-old seedlings under untreated conditions. We developed ISDH-seq (in situ DNase I hypersensitivity sequencing) and compared its performance with DNase-seq and ATAC-seq on the same batch of seedling tissues (Biological replicates n=2).