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Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine

Booth, Michael J ; Ost, Tobias W B ; Beraldi, Dario ; Bell, Neil M ; Branco, Miguel R ; Reik, Wolf ; Balasubramanian, Shankar

Nature protocols, 2013-10, Vol.8 (10), p.1841-1851 [Periódico revisado por pares]

England: Nature Publishing Group

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  • Título:
    Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine
  • Autor: Booth, Michael J ; Ost, Tobias W B ; Beraldi, Dario ; Bell, Neil M ; Branco, Miguel R ; Reik, Wolf ; Balasubramanian, Shankar
  • Assuntos: 5-Methylcytosine - chemistry ; Analysis ; Cytosine - analogs & derivatives ; Cytosine - chemistry ; DNA Methylation ; Enzymes ; Epigenetic inheritance ; Genetic aspects ; Genomes ; Mammals ; Methods ; Methylation ; Oxidation ; Oxidation-Reduction ; Physiological aspects ; Sequence Analysis, DNA - methods
  • É parte de: Nature protocols, 2013-10, Vol.8 (10), p.1841-1851
  • Notas: ObjectType-Article-1
    SourceType-Scholarly Journals-1
    ObjectType-Feature-2
    content type line 23
    AUTHOR CONTRIBUTIONS M.J.B. and S.B. invented the oxBS-seq method. M.J.B. and T.W.B.O. designed the experiments with contributions from all authors. M.J.B., T.W.B.O. and N.M.B. prepared samples and conducted experimental work. D.B. performed data analysis, interpretation and simulations. M.J.B. and T.W.B.O. drafted the manuscript and all authors contributed to writing, reviewing and editing the manuscript.
  • Descrição: 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.
  • Editor: England: Nature Publishing Group
  • Idioma: Inglês
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