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Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine

Nature Protocols volume 7, pages 2159–2170 (2012)Cite this article

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Abstract

A complete understanding of the potential function of 5-hydroxymethylcytosine (5-hmC), a DNA cytosine modification in mammalian cells, requires an accurate single-base resolution sequencing method. Here we describe a modified bisulfite-sequencing method, Tet-assisted bisulfite sequencing (TAB-seq), which can identify 5-hmC at single-base resolution, as well as determine its abundance at each modification site. This protocol involves β-glucosyltransferase (β-GT)-mediated protection of 5-hmC (glucosylation) and recombinant mouse Tet1(mTet1)-mediated oxidation of 5-methylcytosine (5-mC) to 5-carboxylcytosine (5-caC). After the subsequent bisulfite treatment and PCR amplification, both cytosine and 5-caC (derived from 5-mC) are converted to thymine (T), whereas 5-hmC reads as C. The treated genomic DNA is suitable for both whole-genome and locus-specific sequencing. The entire procedure (which does not include data analysis) can be completed in 14 d for whole-genome sequencing or 7 d for locus-specific sequencing.

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Figure 1: Overview of Tet-assisted bisulfite sequencing (TAB-seq).
Figure 2: HPLC analysis of commercial 5-hmdCTP.
Figure 3

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Acknowledgements

This study was supported by the US National Institutes of Health (GM071440 and HG006827 to C.H., U01 ES017166 to B.R., NS079625 and HD073162 to P.J.), a Catalyst Award (to C.H.) from the Chicago Biomedical Consortium with support from the Searle Funds at The Chicago Community Trust, the Ludwig Institute for Cancer Research (to B.R.) and the Emory Genetics Discovery Fund (to P.J.).

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Authors and Affiliations

  1. Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, Chicago, Illinois, USA

    Miao Yu, Chun-Xiao Song & Chuan He

  2. Department of Cellular and Molecular Medicine, San Diego School of Medicine,

    Gary C Hon & Bing Ren

  3. Ludwig Institute for Cancer Research, University of California, La Jolla, California, USA

    Gary C Hon & Bing Ren

  4. Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia, USA

    Keith E Szulwach & Peng Jin

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  1. Miao Yu
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  4. Chun-Xiao Song
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Contributions

M.Y., C.-X.S. and C.H. conceived the original idea. M.Y., C.-X.S. and C.H. designed the experiment with the help from B.R. and P.J.; M.Y. performed treatment of genomic DNA; M.Y., G.C.H. and K.E.S. performed locus-specific sequencing; and G.C.H. and K.E.S. performed genome-wide sequencing. M.Y. and C.H. drafted the manuscript, and all the authors participated in writing and editing the manuscript.

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Correspondence to Chuan He.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Note 1

Sequence of 5hmC spike-in control (PDF 213 kb)

Supplementary Note 2

Insertion Sequence of mTet1. The mouse TET1 (1367-2039) gene with one flag tag was cloned into the insect cell expression plasmid pFastBac Dual (Invitrogen, cat. 10712-024). The restriction enzyme cutting sites are BssHII and NotI. (PDF 199 kb)

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Yu, M., Hon, G., Szulwach, K. et al. Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine. Nat Protoc 7, 2159–2170 (2012). https://doi.org/10.1038/nprot.2012.137

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