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High-Resolution Analysis of 5-Hydroxymethylcytosine by TET-Assisted Bisulfite Sequencing

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DNA Modifications

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2198))

Abstract

DNA cytosine modification is an important epigenetic mechanism that serves critical functions in a variety of biological processes in development and disease. 5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the two most common epigenetic marks found in the mammalian genome. 5hmC is generated from 5mC by the ten-eleven translocation (TET) family of dioxygenase enzymes. This modification can reach substantial levels in certain cell types such as embryonic stem cells and neurons. Standard bisulfite sequencing techniques cannot distinguish between 5mC and 5hmC. Therefore, the method of TET-assisted bisulfite sequencing has been developed for detecting 5hmC specifically. The method is based on protection of 5hmC by glycosylation followed by complete oxidation of both 5mC and 5fC to 5caC, which converts to uracil after bisulfite treatment leaving only 5hmC remaining as a cytosine signal after PCR and sequencing. The method requires a highly active TET protein for the conversion steps. Here, we present an efficient TET protein purification method and a streamlined TAB-sequencing protocol for 5hmC analysis at single base resolution.

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Author information

Author notes

  1. Zhijun Huang and Yingying Meng contributed equally to this work.

Authors and Affiliations

  1. Center for Epigenetics, Van Andel Institute, Grand Rapids, MI, USA

    Zhijun Huang, Yingying Meng, Piroska E. Szabó & Gerd P. Pfeifer

  2. Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Rahul M. Kohli

Authors
  1. Zhijun Huang
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  2. Yingying Meng
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  3. Piroska E. Szabó
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  4. Rahul M. Kohli
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  5. Gerd P. Pfeifer
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Corresponding author

Correspondence to Gerd P. Pfeifer .

Editor information

Editors and Affiliations

  1. Centre for Biomolecular Sciences, University Park, University of Nottingham, Nottingham, UK

    Alexey Ruzov

  2. School of Life Sciences, Queen’s Medical Centre, University of Nottingham, Nottingham, UK

    Martin Gering

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© 2021 The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Huang, Z., Meng, Y., Szabó, P.E., Kohli, R.M., Pfeifer, G.P. (2021). High-Resolution Analysis of 5-Hydroxymethylcytosine by TET-Assisted Bisulfite Sequencing. In: Ruzov, A., Gering, M. (eds) DNA Modifications. Methods in Molecular Biology, vol 2198. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0876-0_25

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  • DOI: https://doi.org/10.1007/978-1-0716-0876-0_25

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0875-3

  • Online ISBN: 978-1-0716-0876-0

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