Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine

Journal name:
Nature Protocols
Year published:
Published online


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.

At a glance


  1. Overview of Tet-assisted bisulfite sequencing (TAB-seq).
    Figure 1: Overview of Tet-assisted bisulfite sequencing (TAB-seq).

    5-hmC is protected specifically by β-GT to generate 5-gmC, followed by oxidation of 5-mC to 5-caC by mTet1. Only 5-gmC is read as C after bisulfite treatment and PCR amplification.

  2. HPLC analysis of commercial 5-hmdCTP.
    Figure 2: HPLC analysis of commercial 5-hmdCTP.

    The commercial 5-hmdCTP contains 4–5% of dCTP. mAU, milliabsorbance units.

  3. Workflow for TAB-seq.
    Figure 3: Workflow for TAB-seq.


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Author information


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

    • Gary C Hon &
    • Bing Ren
  3. Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia, USA.

    • Keith E Szulwach &
    • Peng Jin


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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The authors declare no competing financial interests.

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Supplementary information

PDF files

  1. Supplementary Note 1 (213K)

    Sequence of 5hmC spike-in control

  2. Supplementary Note 2 (199K)

    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.

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