Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine

• 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
• Journal Title: Nature Protocols
• Volume: Volume 7, Number 12
• Publisher: Nature Research (part of Springer Nature) | 2012-12-01, Pages 2159-2170
• Type of Work: Article | Post-print: After Peer Review
• Publisher DOI: http://doi.org/10.1038/nprot.2012.137
• Subjects: Chemistry, Biochemistry; Biology, Genetics; Biophysics, Medical
• Keywords: Science & Technology; Life Sciences & Biomedicine; Biochemical Research Methods; Biochemistry & Molecular Biology; EMBRYONIC STEM-CELLS; DNA METHYLATION; BETA-GLUCOSYLTRANSFERASE; DEOXYRIBONUCLEIC-ACID; BASE-RESOLUTION; MAMMALIAN DNA; THYMINE DNA; 5-METHYLCYTOSINE; GENOME; 5-CARBOXYLCYTOSINE

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.