Characterization of the Nucleosome Landscape by Micrococcal Nuclease-Sequencing (MNase-seq)

  • Wieteke Anna Maria Hoeijmakers
  • Richárd Bártfai
Part of the Methods in Molecular Biology book series (MIMB, volume 1689)


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 words

Micrococcal 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.


  1. 1.
    Lieleg C, Krietenstein N, Walker M, Korber P (2015) Nucleosome positioning in yeasts: methods, maps, and mechanisms. Chromosoma 124:131–151CrossRefPubMedGoogle Scholar
  2. 2.
    Hughes AL, Rando OJ (2014) Mechanisms underlying nucleosome positioning in vivo. Annu Rev Biophys 43:41–63CrossRefPubMedGoogle Scholar
  3. 3.
    Clark RJ, Felsenfeld G (1971) Structure of chromatin. Nat New Biol 229:101–106CrossRefPubMedGoogle Scholar
  4. 4.
    Heins JN, Suriano JR, Taniuchi H, Anfinsen CB (1967) Characterization of a nuclease produced by Staphylococcus aureus. J Biol Chem 242:1016–1020PubMedGoogle Scholar
  5. 5.
    Cui K, Zhao K (2012) Genome-wide approaches to determining nucleosome occupancy in metazoans using MNase-Seq. Methods Mol Biol 833:413–419CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Platt JL, Kent NA, Harwood AJ, Kimmel AR (2013) Analysis of chromatin organization by deep sequencing technologies. Methods Mol Biol 983:173–183CrossRefPubMedGoogle Scholar
  7. 7.
    Ishii H, Kadonaga JT, Ren B (2015) MPE-seq, a new method for the genome-wide analysis of chromatin structure. Proc Natl Acad Sci U S A 112:E3457–E3465CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Rhee HS, Bataille AR, Zhang L, Pugh BF (2014) Subnucleosomal structures and nucleosome asymmetry across a genome. Cell 159:1377–1388CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Brogaard K, Xi L, Wang JP, Widom J (2012) A map of nucleosome positions in yeast at base-pair resolution. Nature 486:496–501PubMedPubMedCentralGoogle Scholar
  10. 10.
    Allan J, Fraser RM, Owen-Hughes T, Keszenman-Pereyra D (2012) Micrococcal nuclease does not substantially bias nucleosome mapping. J Mol Biol 417:152–164CrossRefPubMedGoogle Scholar
  11. 11.
    Kensche PR, Hoeijmakers WA, Toenhake CG, Bras M, Chappell L, Berriman M, Bartfai R (2016) The nucleosome landscape of Plasmodium falciparum reveals chromatin architecture and dynamics of regulatory sequences. Nucleic Acids Res 44:2110–2124CrossRefPubMedGoogle Scholar
  12. 12.
    Orlando V, Strutt H, Paro R (1997) Analysis of chromatin structure by in vivo formaldehyde cross-linking. Methods 11:205–214CrossRefPubMedGoogle Scholar
  13. 13.
    Mieczkowski J, Cook A, Bowman SK, Mueller B, Alver BH, Kundu S, Deaton AM, Urban JA, Larschan E, Park PJ, Kingston RE, Tolstorukov MY (2016) MNase titration reveals differences between nucleosome occupancy and chromatin accessibility. Nat Commun 7:11485CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Rizzo JM, Bard JE, Buck MJ (2012) Standardized collection of MNase-seq experiments enables unbiased dataset comparisons. BMC Mol Biol 13:15CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Horz W, Altenburger W (1981) Sequence specific cleavage of DNA by micrococcal nuclease. Nucleic Acids Res 9:2643–2658CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Kaplan N, Hughes TR, Lieb JD, Widom J, Segal E (2010) Contribution of histone sequence preferences to nucleosome organization: proposed definitions and methodology. Genome Biol 11:140CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Nikitina T, Wang D, Gomberg M, Grigoryev SA, Zhurkin VB (2013) Combined micrococcal nuclease and exonuclease III digestion reveals precise positions of the nucleosome core/linker junctions: implications for high-resolution nucleosome mapping. J Mol Biol 425:1946–1960CrossRefPubMedGoogle Scholar
  18. 18.
    Meyer CA, Liu XS (2014) Identifying and mitigating bias in next-generation sequencing methods for chromatin biology. Nat Rev Genet 15:709–721CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Oyola SO, Otto TD, Gu Y, Maslen G, Manske M, Campino S, Turner DJ, Macinnis B, Kwiatkowski DP, Swerdlow HP, Quail MA (2012) Optimizing Illumina next-generation sequencing library preparation for extremely AT-biased genomes. BMC Genomics 13:1CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Hoeijmakers WA, Bartfai R, Francoijs KJ, Stunnenberg HG (2010) Linear amplification for deep sequencing. Nat Protoc 6:1026–1036CrossRefGoogle Scholar
  21. 21.
    Kivioja T, Vaharautio A, Karlsson K, Bonke M, Enge M, Linnarsson S, Taipale J (2012) Counting absolute numbers of molecules using unique molecular identifiers. Nat Methods 9:72–74CrossRefGoogle Scholar
  22. 22.
    Quintales L, Vazquez E, Antequera F (2014) Comparative analysis of methods for genome-wide nucleosome cartography. Brief Bioinform 16:576–587CrossRefPubMedGoogle Scholar
  23. 23.
    Teif VB (2015) Nucleosome positioning: resources and tools online. Brief Bioinform 17:745–757CrossRefPubMedGoogle Scholar
  24. 24.
    Henikoff JG, Belsky JA, Krassovsky K, MacAlpine DM, Henikoff S (2011) Epigenome characterization at single base-pair resolution. Proc Natl Acad Sci U S A 108:18318–18323CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Hoeijmakers WA, Bartfai R, Francoijs KJ, Stunnenberg HG (2011) Linear amplification for deep sequencing. Nat Protoc 6:1026–1036CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Department of Molecular BiologyRadboud Institute for Molecular Life Sciences, Faculty of Science, Radboud UniversityNijmegenThe Netherlands

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