title: 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 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.
authors.affiliation: Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA.
authors.forename: Miao
authors.initials: M
authors.lastname: Yu
authors.forename: Gary C
authors.initials: GC
authors.lastname: Hon
authors.forename: Keith E
authors.initials: KE
authors.lastname: Szulwach
authors.forename: Chun-Xiao
authors.initials: CX
authors.lastname: Song
authors.forename: Peng
authors.initials: P
authors.lastname: Jin
authors.forename: Bing
authors.initials: B
authors.lastname: Ren
authors.forename: Chuan
authors.initials: C
authors.lastname: He
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003596:Cytosine / Q000031:analogs & derivatives* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005964:Glucosyltransferases / Q000378:metabolism*; D051379:Mice; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D013447:Sulfites; C540839:TET1 protein, mouse; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; D005964:Glucosyltransferases; C042345:hydrogen sulfite