Important considerations in the design of MNase-seq experiments include extent of chromatin crosslinking and level of digestion. Traditionally, chromatin accessibility experiments have been conducted with formaldehyde as a crosslinking agent to capture in vivo protein-nucleic acid and protein-protein interactions [51]. It has been observed that in the absence of crosslinking, nucleosome organization can change during regular chromatin preparation steps and thus use of formaldehyde is recommended for accurate characterization of chromatin structure [31]. Also, MNase has been shown to have a high degree of AT-cleavage specificity in limiting enzyme concentrations [52–54] and comparisons between different experiments will vary for technical reasons unless MNase digestion conditions are tightly controlled [55–57]. MNase titration experiments specifically support differential digestion susceptibility of certain nucleosome classes, with nucleosomes within promoter and ‘nucleosome-free’ regions being highly sensitive [50, 58, 59]. Thus, it has been suggested that combination of templates from different levels of MNase digestion may alleviate biased sampling of mononucleosome populations [58].
