title: Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution.
abstract: Recently, the cytosine modifications 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) were found to exist in the genomic deoxyribonucleic acid (DNA) of a wide range of mammalian cell types. It is now important to understand their role in normal biological function and disease. Here we introduce reduced bisulfite sequencing (redBS-Seq), a quantitative method to decode 5fC in DNA at single-base resolution, based on a selective chemical reduction of 5fC to 5hmC followed by bisulfite treatment. After extensive validation on synthetic and genomic DNA, we combined redBS-Seq and oxidative bisulfite sequencing (oxBS-Seq) to generate the first combined genomic map of 5-methylcytosine, 5hmC and 5fC in mouse embryonic stem cells. Our experiments revealed that in certain genomic locations 5fC is present at comparable levels to 5hmC and 5mC. The combination of these chemical methods can quantify and precisely map these three cytosine derivatives in the genome and will help provide insights into their function.
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, UK.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Booth
authors.affiliation: Cancer Research UK, Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Marsico
authors.affiliation: Cancer Research UK, Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK.
authors.forename: Martin
authors.initials: M
authors.lastname: Bachman
authors.affiliation: Cancer Research UK, Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK.
authors.forename: Dario
authors.initials: D
authors.lastname: Beraldi
authors.affiliation: 1] Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, UK [2] Cancer Research UK, Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK.
authors.forename: Shankar
authors.initials: S
authors.lastname: Balasubramanian
mesh_terms: D000818:Animals; D003596:Cytosine / Q000031:analogs & derivatives* / Q000032:analysis; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D053595:Embryonic Stem Cells / Q000378:metabolism; D051379:Mice; D013447:Sulfites
chemical_list: C560973:5-formylcytosine; D013447:Sulfites; D003596:Cytosine; D004247:DNA; C042345:hydrogen sulfite
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