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Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine.

ABSTRACT: To uncover the function of and interplay between the mammalian cytosine modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), new techniques and advances in current technology are needed. To this end, we have developed oxidative bisulfite sequencing (oxBS-seq), which can quantitatively locate 5mC and 5hmC marks at single-base resolution in genomic DNA. In bisulfite sequencing (BS-seq), both 5mC and 5hmC are read as cytosines and thus cannot be discriminated; however, in oxBS-seq, specific oxidation of 5hmC to 5-formylcytosine (5fC) and conversion of the newly formed 5fC to uracil (under bisulfite conditions) means that 5hmC can be discriminated from 5mC. A positive readout of actual 5mC is gained from a single oxBS-seq run, and 5hmC levels are inferred by comparison with a BS-seq run. Here we describe an optimized second-generation protocol that can be completed in 2 d.

SUBMITTER: Booth MJ

PROVIDER: S-EPMC3919000 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine.

Booth Michael J MJ Ost Tobias W B TW Beraldi Dario D Bell Neil M NM Branco Miguel R MR Reik Wolf W Balasubramanian Shankar S

Nature protocols 20130905 10

To uncover the function of and interplay between the mammalian cytosine modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), new techniques and advances in current technology are needed. To this end, we have developed oxidative bisulfite sequencing (oxBS-seq), which can quantitatively locate 5mC and 5hmC marks at single-base resolution in genomic DNA. In bisulfite sequencing (BS-seq), both 5mC and 5hmC are read as cytosines and thus cannot be discriminated; however, in oxBS-s ...[more]

PMID: 24008380

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OxyBS: estimation of 5-methylcytosine and 5-hydroxymethylcytosine from tandem-treated oxidative bisulfite and bisulfite DNA.

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