Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine

• Type of Material: Article
• Authors: Miao Yu, University of Chicago; Gary C. Hon, University of California San Diego; Keith E. Szulwach, Emory University; Chun-Xiao Song, University of Chicago; Peng Jin, Emory University; Bing Ren, Emory University; Chuan He, University of Chicago
• Language: English
• Date: 2012-12-01
• Publisher: Nature Research (part of Springer Nature)
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2012 Nature America, Inc.
• Final Published Version (URL): http://dx.doi.org/10.1038/nprot.2012.137
• Title of Journal or Parent Work: Nature Protocols
• ISSN: 1754-2189
• Volume: 7
• Issue: 12
• Start Page: 2159
• End Page: 2170
• Grant/Funding Information: 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.).
• Keywords: EMBRYONIC STEM-CELLS; DEOXYRIBONUCLEIC-ACID; Life Sciences & Biomedicine; 5-CARBOXYLCYTOSINE; Biochemical Research Methods; BASE-RESOLUTION; Science & Technology; Biochemistry & Molecular Biology; DNA METHYLATION; 5-METHYLCYTOSINE; THYMINE DNA; MAMMALIAN DNA; GENOME; BETA-GLUCOSYLTRANSFERASE
• Research Categories: Chemistry, Biochemistry; Biophysics, Medical; Biology, Genetics

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.