title: Transcriptome-Wide Detection of 5-Methylcytosine by Bisulfite Sequencing.
abstract: While low-throughput RNA bisulfite sequencing is the method of choice to assess the methylation status of specific cytosines in candidate RNAs, the combination of bisulfite treatment of RNA with today's high-throughput sequencing techniques opens the door to methylation studies at nucleotide resolution on a transcriptome-wide scale. Below we describe a protocol for the transcriptome-wide analysis of total or fractionated poly(A)RNA in cells and tissues. Although the nature of the bisulfite sequencing protocol makes it comparably easy to translate from a low to a high-throughput approach, several critical points require attention before starting such a project. We describe a step-by-step protocol for planning and performing the experiment and analyzing the data.
authors.affiliation: Division of Molecular Biology, Biocenter, Medical University of Innsbruck, Innrain 80-82, Innsbruck, 6020, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Amort
authors.affiliation: Zymo Research Corp, Irvine, CA, USA.
authors.forename: Xueguang
authors.initials: X
authors.lastname: Sun
authors.affiliation: Zymo Research Corp, Irvine, CA, USA.
authors.forename: Daria
authors.initials: D
authors.lastname: Khokhlova-Cubberley
authors.affiliation: Division of Molecular Biology, Biocenter, Medical University of Innsbruck, Innrain 80-82, Innsbruck, 6020, Austria. alexandra.lusser@i-med.ac.at.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Lusser
mesh_terms: D044503:5-Methylcytosine / Q000737:chemistry*; D019295:Computational Biology / Q000379:methods; D020869:Gene Expression Profiling; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing; D008745:Methylation; D012313:RNA / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D059467:Transcriptome
chemical_list: D012333:RNA, Messenger; D012313:RNA; D044503:5-Methylcytosine
keywords: 5-Methylcytosine; Bisulfite sequencing; High throughput; Next-generation sequencing; RNA methylation; Transcriptome