期刊
NATURE PROTOCOLS
卷 7, 期 12, 页码 2159-2170出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2012.137
关键词
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资金
- US National Institutes of Health [GM071440, HG006827, U01 ES017166, NS079625, HD073162]
- Chicago Biomedical Consortium
- Chicago Community Trust
- Ludwig Institute for Cancer Research
- Emory Genetics Discovery Fund
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 beta-glucosyltransferase (beta-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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