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Single Base Resolution Analysis of 5-Methylcytosine and 5-Hydroxymethylcytosine by RRBS and TAB-RRBS

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Cancer Epigenetics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1238))

Abstract

Sodium bisulfite-assisted deamination of cytosine forms the basis for conducting single base resolution analysis of 5-methylcytosine in DNA. The TET family of proteins represents a group of enzymes that can oxidize 5-methylcytosine to 5-hydroxymethylcytosine. A modification of the bisulfite-based DNA methylation mapping technique employs TET1-mediated oxidation of 5-methylcytosine (TET-assisted bisulfite sequencing) for single base analysis of 5-hydroxymethylcytosine. Whole genome analysis of cytosine modifications with bisulfite sequencing techniques still is challenging and expensive. Reduced representation bisulfite sequencing (RRBS) has been used to limit the complexity of the analysis to mostly CpG-rich genomic fragments flanked by restriction enzyme cleavage sites, for example MspI (5′CCGG). In this chapter, we describe detailed methods used in our laboratory for analysis of 5-methylcytosine and 5-hydroxymethylcytosine combined (RRBS) and for specific analysis of 5-hydroxymethylcytosine (TAB-RRBS).

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Acknowledgements

This work was supported by NIH grants CA084469, CA160965, and MH094599 (recipient G.P.P.).

Conflict of interest statement: G.P.P. is a paid consultant of Zymo Research; Irvine, CA.

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Authors and Affiliations

  1. Department of Cancer Biology, Beckman Research Institute, City of Hope, Duarte, CA, USA

    Maria A. Hahn & Gerd P. Pfeifer

  2. Department of Information Sciences, Beckman Research Institute, City of Hope, Duarte, CA, USA

    Arthur X. Li

  3. Department of Molecular and Cellular Biology, Beckman Research Institute, City of Hope, Duarte, CA, USA

    Xiwei Wu

  4. Beckman Research Institute, City of Hope, 1500 East Duarte Road, Duarte, CA, 91010, USA

    Gerd P. Pfeifer

Authors
  1. Maria A. Hahn
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  2. Arthur X. Li
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  3. Xiwei Wu
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  4. Gerd P. Pfeifer
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Corresponding author

Correspondence to Gerd P. Pfeifer .

Editor information

Editors and Affiliations

  1. National Institutes of Health, Rockville, Maryland, USA

    Mukesh Verma

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Hahn, M.A., Li, A.X., Wu, X., Pfeifer, G.P. (2015). Single Base Resolution Analysis of 5-Methylcytosine and 5-Hydroxymethylcytosine by RRBS and TAB-RRBS. In: Verma, M. (eds) Cancer Epigenetics. Methods in Molecular Biology, vol 1238. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1804-1_14

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  • DOI: https://doi.org/10.1007/978-1-4939-1804-1_14

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1803-4

  • Online ISBN: 978-1-4939-1804-1

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