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Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine.


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 ?-glucosyltransferase (?-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.

SUBMITTER: Yu M 

PROVIDER: S-EPMC3641661 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine.

Yu Miao M   Hon Gary C GC   Szulwach Keith E KE   Song Chun-Xiao CX   Jin Peng P   Ren Bing B   He Chuan C  

Nature protocols 20121129 12


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 (gluc  ...[more]

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