Transcriptome-Wide Detection of 5-Methylcytosine by Bisulfite Sequencing

  • Thomas Amort
  • Xueguang Sun
  • Daria Khokhlova-Cubberley
  • Alexandra LusserEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1562)


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.

Key words

Bisulfite sequencing RNA methylation 5-Methylcytosine High throughput Transcriptome Next-generation sequencing 



Funding in A.L’s lab is provided by the Austrian Science Fund (FWF): P27024-BBL.


  1. 1.
    Grosjean H (2009) Nucleic acids are not boring long polymers of only four types of nucleotides: a guided tour. In: Grosjean H (ed) DNA and RNA modification enzymes: structure, mechanism, function and evolution. LandesBioscience, AustinGoogle Scholar
  2. 2.
    Dubin DT, Taylor RH (1975) The methylation state of poly A-containing messenger RNA from cultured hamster cells. Nucleic Acids Res 2:1653–1668CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Salditt-Georgieff M, Jelinek W, Darnell JE, Furuichi Y, Morgan M, Shatkin A (1976) Methyl labeling of HeLa cell hnRNA: a comparison with mRNA. Cell 7:227–237CrossRefPubMedGoogle Scholar
  4. 4.
    Squires JE, Patel HR, Nousch M, Sibbritt T, Humphreys DT, Parker BJ, Suter CM, Preiss T (2012) Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA. Nucleic Acids Res 40:5023–5033. doi: 10.1093/nar/gks144 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Hayatsu H, Wataya Y, Kai K, Iida S (1970) Reaction of sodium bisulfite with uracil, cytosine, and their derivatives. Biochemistry 9:2858–2865CrossRefPubMedGoogle Scholar
  6. 6.
    Shapiro R, Servis RE, Welcher M (1970) Reactions of uracil and cytosine derivatives with sodium bisulfite. J Am Chem Soc 92(2):422–424CrossRefGoogle Scholar
  7. 7.
    Hayatsu H, Shiragami M (1979) Reaction of bisulfite with the 5-hydroxymethyl group in pyrimidines and in phage DNAs. Biochemistry 18:632–637CrossRefPubMedGoogle Scholar
  8. 8.
    Amort T, Soulière MF, Wille A, Jia X-Y, Fiegl H, Wörle H, Micura R, Lusser A (2013) Long non-coding RNAs as targets for cytosine methylation. RNA Biol 10:1003–1008. doi: 10.4161/rna.24454 CrossRefPubMedGoogle Scholar
  9. 9.
    Rieder D, Amort T, Kugler E, Lusser A, Trajanoski Z (2015) meRanTK: methylated RNA analysis ToolKit. Bioinformatics. doi: 10.1093/bioinformatics/btv647
  10. 10.
    Schaefer M, Pollex T, Hanna K, Lyko F (2009) RNA cytosine methylation analysis by bisulfite sequencing. Nucleic Acids Res 37:e12. doi: 10.1093/nar/gkn954 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Thomas Amort
    • 1
  • Xueguang Sun
    • 2
  • Daria Khokhlova-Cubberley
    • 2
  • Alexandra Lusser
    • 1
    Email author
  1. 1.Division of Molecular Biology, BiocenterMedical University of InnsbruckInnsbruckAustria
  2. 2.Zymo Research CorpIrvineUSA

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