Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine

Miao Yu, Gary C. Hon, Keith E. Szulwach, Chun Xiao Song, Peng Jin, Bing Ren, Chuan He

Research output: Contribution to journalArticle

147 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)2159-2170
Number of pages12
JournalNature Protocols
Volume7
Issue number12
DOIs
StatePublished - Dec 2012

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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    Yu, M., Hon, G. C., Szulwach, K. E., Song, C. X., Jin, P., Ren, B., & He, C. (2012). Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine. Nature Protocols, 7(12), 2159-2170. https://doi.org/10.1038/nprot.2012.137