Micrococcal nuclease sequencing is the state-of-the-art method for determining chromatin structure and nucleosome positioning. Data analysis is complex due to the AT-dependent sequence bias of the endonuclease and the requirement for high sequencing depth. Here, we present the nucleosome-based MNase accessibility (nucMACC) pipeline unveiling the regulatory chromatin landscape by measuring nucleosome accessibility and stability. The nucMACC pipeline represents a systematic and genome-wide approach for detecting unstable (“fragile”) nucleosomes. We have characterized the regulatory nucleosome landscape in Drosophila melanogaster, Saccharomyces cerevisiae, and mammals. Two functionally distinct sets of promoters were characterized, one associated with an unstable nucleosome and the other being nucleosome depleted. We show that unstable nucleosomes present intermediate states of nucleosome remodeling, preparing inducible genes for transcriptional activation in response to stimuli or stress. The presence of unstable nucleosomes correlates with RNA polymerase II proximal pausing. The nucMACC pipeline offers unparalleled precision and depth in nucleosome research and is a valuable tool for future nucleosome studies.