Characterization of the Nucleosome Landscape by Micrococcal Nuclease-Sequencing (MNase-seq)
MNase-seq allows the genome-wide examination of the nucleosome landscape by determination of nucleosome positioning and occupancy. Typically, native or formaldehyde fixed chromatin is subjected to digestion by micrococcal nuclease (MNase), which degrades linker DNA and yields mainly mono-nucleosomes. The resulting material can be processed directly or can be subjected to an optional chromatin immunoprecipitation step (MNase-ChIP-seq). De-crosslinked and purified DNA is then subjected to next-generation sequencing. The protocol presented here has been tailored for the analysis of nucleosome landscape in the malaria parasite, Plasmodium falciparum, but most steps are directly applicable to other cell types. We also discuss general considerations for experimental design and computational analysis, which are crucial for accurate investigation of the nucleosome landscape.
Key wordsMicrococcal nuclease (MNase) MNase-seq MNase-ChIP-seq Nucleosome landscape Nucleosome positioning Nucleosome occupancy AT-rich DNA Plasmodium falciparum
The research leading to this protocol has received funding from The Netherlands Organization for Scientific Research (NWO-Vidi 864.11.007 to R.B.) and The National Institutes of Health (EuPathDB-Driving Biological Project subaward # 553539 to R.B.). We would like to acknowledge Dr. Kensche for valuable input and discussions concerning data analysis and Christa Toenhake for proofreading of the manuscript. Furthermore, we would like to thank our colleagues at the Department of Molecular Biology, the Department of Molecular Developmental Biology, and the Department of Medical Microbiology of Radboud University and St. Radboud UMC for support and advice.