Mammalian chromatin is dynamic, and contains active DNA regulatory elements that lead to its accessibility to transcription factors, and gene expression. Here, we describe a novel accessible chromatin mapping methodology using Nicking Enzyme assisted sequencing (NicE-seq) for integrative epigenomic analysis (Ponnaluri et al., 2017). Universal NicE-seq (UniNicE-seq) captures open chromatin sites in both fixed and unfixed cells and reveals the genomic location of accessible/open chromatin sites (OCS) and transcription factor occupancy at single nucleotide resolution, with as few as 25 cells. OCS were coincident with DNaseI hypersensitivity (DNase-seq, Boyle et al., 2008) and ATAC-seq (Buenrostro et al., 2013) sites at a low sequencing burden of ~20 million reads. Open chromatin sites correlated with RNA pol II occupancy and transcriptionally active chromatin marks, while displaying a pattern contrasting to CpG methylation. We demonstrate the general applicability of the accessible chromatin profiling protocol for a variety of plant and mammalian cells, as well as with frozen 5-10 µM human fetal and adult tissue sections. In summary, UniNicE-seq allows a simpler and robust means to study the accessible chromatin structure of the mammalian genome, revealing the open chromatin landscape in a cellular and developmental context.