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Myogenesis pp 43-54 | Cite as

Chromatin Immunoprecipitation in Skeletal Myoblasts

  • Vinay Kumar Rao
  • Shilpa Rani Shankar
  • Reshma TanejaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1889)

Abstract

Chromatin immunoprecipitation (ChIP) is a powerful and sensitive technique that is widely used to study DNA-protein interactions. It enables an unbiased genome-wide analysis of transcriptional changes during several biological processes including cellular differentiation. Here, we describe a step-by-step protocol to identify histone modifications, transcription factor, and co-factor binding to chromatin in skeletal myoblasts. We discuss critical steps during cell harvesting, sonication, and immunoprecipitation and provide notes to evade common pitfalls.

Key words

Myoblasts Chromatin modifiers Histone modifications Epigenetics Transcription 

Notes

Acknowledgments

Work in the R.T. laboratory is supported by grants from the National Medical Research Council.

References

  1. 1.
    Kassar-Duchossoy L, Giacone E, Gayraud-Morel B, Jory A, Gomès D, Tajbakhsh S (2005) Pax3/Pax7 mark a novel population of primitive myogenic cells during development. Genes Dev 19:1426–1431.  https://doi.org/10.1101/gad.345505CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Bentzinger CF, Wang YX, Rudnicki MA (2012) Building muscle: molecular regulation of myogenesis. Cold Spring Harb Perspect Biol 4.  https://doi.org/10.1101/cshperspect.a008342CrossRefGoogle Scholar
  3. 3.
    Bharathy N, Ling BMT, Taneja R (2013) Epigenetic regulation of skeletal muscle development and differentiation. Subcell Biochem 61:139–150.  https://doi.org/10.1007/978-94-007-4525-4_7CrossRefPubMedGoogle Scholar
  4. 4.
    McKinsey TA, Zhang CL, Olson EN (2001) Control of muscle development by dueling HATs and HDACs. Curr Opin Genet Dev 11:497–504CrossRefGoogle Scholar
  5. 5.
    Asp P, Blum R, Vethantham V, Parisi F, Micsinai M, Cheng J, Bowman C, Kluger Y, Dynlacht BD (2011) Genome-wide remodeling of the epigenetic landscape during myogenic differentiation. Proc Natl Acad Sci U S A 108:E149–E158.  https://doi.org/10.1073/pnas.1102223108CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Fittipaldi R, Caretti G (2012) Tackling skeletal muscle cells epigenome in the next-generation sequencing era. Comp Funct Genomics 2012:979168.  https://doi.org/10.1155/2012/979168CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Cao Y, Yao Z, Sarkar D, Lawrence M, Sanchez GJ, Parker MH, MacQuarrie KL, Davison J, Morgan MT, Ruzzo WL, Gentleman RC, Tapscott SJ (2010) Genome-wide MyoD binding in skeletal muscle cells: a potential for broad cellular reprogramming. Dev Cell 18:662–674.  https://doi.org/10.1016/j.devcel.2010.02.014CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Blum R, Vethantham V, Bowman C, Rudnicki M, Dynlacht BD (2012) Genome-wide identification of enhancers in skeletal muscle: the role of MyoD1. Genes Dev 26:2763–2779.  https://doi.org/10.1101/gad.200113.112CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Lu L, Sun K, Chen X, Zhao Y, Wang L, Zhou L, Sun H, Wang H (2013) Genome-wide survey by ChIP-seq reveals YY1 regulation of lincRNAs in skeletal myogenesis. EMBO J 32:2575–2588.  https://doi.org/10.1038/emboj.2013.182CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Ohkawa Y, Mallappa C, Vallaster CSD, Imbalzano AN (2012) Isolation of nuclei from skeletal muscle satellite cells and myofibers for use in chromatin immunoprecipitation assays. Methods Mol Biol 798:517–530.  https://doi.org/10.1007/978-1-61779-343-1_31CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Nelson JD, Denisenko O, Bomsztyk K (2006) Protocol for the fast chromatin immunoprecipitation (ChIP) method. Nat Protoc 1:179–185.  https://doi.org/10.1038/nprot.2006.27CrossRefPubMedGoogle Scholar
  12. 12.
    Kuo MH, Allis CD (1999) In vivo cross-linking and immunoprecipitation for studying dynamic protein:DNA associations in a chromatin environment. Methods 19:425–433.  https://doi.org/10.1006/meth.1999.0879CrossRefPubMedGoogle Scholar
  13. 13.
    Nelson JD, Denisenko O, Sova P, Bomsztyk K (2006) Fast chromatin immunoprecipitation assay. Nucleic Acids Res 34:e2.  https://doi.org/10.1093/nar/gnj004CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Thorne AW, Myers FA, Hebbes TR (2004) Native chromatin immunoprecipitation. Methods Mol Biol 287:21–44.  https://doi.org/10.1385/1-59259-828-5:021CrossRefPubMedGoogle Scholar
  15. 15.
    Schoppee Bortz PD, Wamhoff BR (2011) Chromatin immunoprecipitation (ChIP): revisiting the efficacy of sample preparation, sonication, quantification of sheared DNA, and analysis via PCR. PLoS One 6:e26015.  https://doi.org/10.1371/journal.pone.0026015CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Vinay Kumar Rao
    • 1
    • 2
  • Shilpa Rani Shankar
    • 1
  • Reshma Taneja
    • 1
    Email author
  1. 1.Department of Physiology, Yong Loo Lin School of MedicineNational University of SingaporeSingaporeSingapore
  2. 2.Temasek Life Sciences LaboratoryNational University of SingaporeSingaporeSingapore

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