Fig. 4: Proposed model of the effect of fixation and long-term storage on MNase accessibility and occupancy signal.

Conceptual model of the combined effects of fixation and storage conditions on MNase occupancy signal. From top to bottom, (Accessibility) Applied to lightly fixed chromatin, the MNase enzyme (depicted in orange) cleaves DNA adjacent to the nucleosome and resects unbound DNA, thus releasing nucleosome-bound DNA. Applied to heavily fixed chromatin, the MNase enzyme’s access to unbound DNA is modulated by chromatin accessibility, thus reducing the release of nucleosome-bound DNA only from within the most accessible chromatin regions. Applied to archivally fixed chromatin, prolonged MNase digestion is required to release sufficient DNA for sequencing from the heavily fixed chromatin within intact whole specimens stored for months to many years. This prolonged digestion preferentially degrades both linker DNA and nucleosome-bound DNA in MNase-accessible regions and releases fragments from relatively inaccessible regions. (Read depth) Relative accessibility of the MNase enzyme alters the read depth pattern observed in the region of euchromatin relative to heterochromatin. (DANPOS profile) DANPOS efficiently detects both relative occupancy value gains and losses resulting from MNase digestion. (Example system) We offer examples of light fixation in both a single (yeast) and multicellular (mouse) system with no storage time, heavy fixation in a single (yeast) cellular system with no storage time and archival fixation in two multicellular vertebrate systems stored for several years (mouse) or up to 117 years (water dragon). (DANPOS peak Heatmaps) For each example system, we show a heatmap of MNase assay significant peak gains and losses (FDR < 0.05, log10Pval < −6) 2 kb on either side of genome-wide transcription start sites pooled across all replicates (three for yeast and mouse, 5 for water dragon). Under light and heavy fixation, the predominant genome-wide signal appears as occupancy gains, while under archival fixation, the predominant genome-wide signal appears as occupancy losses irrespective of storage time. Created with BioRender.com, released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.