However, the cause of differences in MNase-seq output across differential levels of enzymatic digestion is difficult to assess due to the effect of inter-nucleosomal linker length on the recovered signal [55]. MNase digestion simulation experiments have provided evidence that nucleosome configurations with or near long linkers are sampled easier compared to nucleosomes with normal linkers at low levels of MNase digestion and this sampling bias dissipitates with increased levels of enzymatic cleavage (80 or 100% monos) [55]. Comparison of in vivo experimental data of two distinct nucleosome configurations from different MNase-seq technical preparations supports the same conclusions, and underscores the importance of standardized collection of mononucleosomes for accurate and reproducible comparisons [55]. Specifically, extensive (approximately 95 to 100% mononucleosomes) digestion of a standardized initial amount of crosslinked chromatin is considered ideal for comparisons of different MNase-seq experiments, since at that level of digestion all linkers are cut and the recovered signal is not confounded by nucleosome configuration [31, 55].
