Micrococcal nuclease sequencing (MNase-seq) is the current state-of-the-art method for determining the chromatin structure and nucleosome positioning. The analysis of MNase-seq data is complex due to the variability in sample quality, the AT-dependent sequence bias of the endonuclease, and the requirement for high sequencing depth. We have therefore developed an optimized and automated analysis pipeline and provide guidelines for MNase-seq experiments. The nucleosome based MNase accessibility (nucMACC) pipeline provides high-resolution nucleosome positions and associated properties such as nucleosome accessibility and stability. Using the nucMACC pipeline, we characterized the nucleosomal landscape in D. melanogaster and S. cerevisiae. Interestingly, we were able to distinguish between two sets of promoters, one associated with an unstable nucleosome and the other nucleosome depleted. Our data suggest that unstable nucleosomes are intermediate states of nucleosome remodeling that prepare inducible genes for transcriptional activation in response to stimuli or stress by regulating RNA polymerase II proximal pausing.