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5-Methylcytosine Analysis by RNA-BisSeq

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Epitranscriptomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1870))

Abstract

5-Methylcytosine (m5C) is a posttranscriptional RNA modification identified in both stable and highly abundant tRNAs and rRNAs, and in mRNAs. Many known or novel m5C sites have been validated by using advanced high-throughput techniques combined with next-generation sequencing (NGS), especially RNA bisulfite sequencing (RNA-BisSeq). Here we introduce an optimized RNA-BisSeq method by using ACT random hexamers to prime the reverse transcription of bisulfite-treated RNA samples to detect the m5C sites.

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References

  1. Schaefer M (2015) RNA 5-methylcytosine analysis by bisulfite sequencing. Methods Enzymol 560:297–329. https://doi.org/10.1016/bs.mie.2015.03.007

    Article  CAS  PubMed  Google Scholar 

  2. Edelheit S, Schwartz S, Mumbach MR, Wurtzel O, Sorek R (2013) Transcriptome-wide mapping of 5-methylcytidine RNA modifications in bacteria, archaea, and yeast reveals m5C within archaeal mRNAs. PLoS Genet 9(6):e1003602. https://doi.org/10.1371/journal.pgen.1003602

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Hussain S, Aleksic J, Blanco S, Dietmann S, Frye M (2013) Characterizing 5-methylcytosine in the mammalian epitranscriptome. Genome Biol 14(11):215. https://doi.org/10.1186/gb4143

    Article  PubMed  PubMed Central  Google Scholar 

  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(11):5023–5033. https://doi.org/10.1093/nar/gks144

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Hussain S, Sajini AA, Blanco S, Dietmann S, Lombard P, Sugimoto Y, Paramor M, Gleeson JG, Odom DT, Ule J, Frye M (2013) NSun2-mediated cytosine-5 methylation of vault noncoding RNA determines its processing into regulatory small RNAs. Cell Rep 4(2):255–261. https://doi.org/10.1016/j.celrep.2013.06.029

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Meyer KD, Jaffrey SR (2014) The dynamic epitranscriptome: N6-methyladenosine and gene expression control. Nat Rev Mol Cell Biol 15(5):313–326. https://doi.org/10.1038/nrm3785

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Saletore Y, Chen-Kiang S, Mason CE (2013) Novel RNA regulatory mechanisms revealed in the epitranscriptome. RNA Biol 10(3):342–346. https://doi.org/10.4161/rna.23812

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Saletore Y, Meyer K, Korlach J, Vilfan ID, Jaffrey S, Mason CE (2012) The birth of the Epitranscriptome: deciphering the function of RNA modifications. Genome Biol 13(10):175. https://doi.org/10.1186/gb-2012-13-10-175

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Amort T, Rieder D, Wille A, Khokhlova-Cubberley D, Riml C, Trixl L, Jia XY, Micura R, Lusser A (2017) Distinct 5-methylcytosine profiles in poly(A) RNA from mouse embryonic stem cells and brain. Genome Biol 18(1):1. https://doi.org/10.1186/s13059-016-1139-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. David R, Burgess A, Parker B, Li J, Pulsford K, Sibbritt T, Preiss T, Searle IR (2017) Transcriptome-wide mapping of RNA 5-methylcytosine in arabidopsis mRNAs and noncoding RNAs. Plant Cell 29(3):445–460. https://doi.org/10.1105/tpc.16.00751

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Yang X, Yang Y, Sun BF, Chen YS, Xu JW, Lai WY, Li A, Wang X, Bhattarai DP, Xiao W, Sun HY, Zhu Q, Ma HL, Adhikari S, Sun M, Hao YJ, Zhang B, Huang CM, Huang N, Jiang GB, Zhao YL, Wang HL, Sun YP, Yang YG (2017) 5-methylcytosine promotes mRNA export - NSUN2 as the methyltransferase and ALYREF as an m(5)C reader. Cell Res 27(5):606–625. https://doi.org/10.1038/cr.2017.55

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. SA (2010) Fastqc. A quality control tool for high throughput sequence data. http://wwwbioinformaticsbabrahamacuk/projects/fastqc

    Google Scholar 

  13. Martin M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J 17:10–12

    Article  Google Scholar 

  14. Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30(15):2114–2120. https://doi.org/10.1093/bioinformatics/btu170

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Rieder D, Amort T, Kugler E, Lusser A, Trajanoski Z (2016) meRanTK: methylated RNA analysis ToolKit. Bioinformatics 32(5):782–785. https://doi.org/10.1093/bioinformatics/btv647

    Article  CAS  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. CAS Key Laboratory of Genomic and Precision Medicine, College of Futuer Technology, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China

    Yu-Sheng Chen, Hai-Li Ma, Ying Yang, Bao-Fa Sun & Yun-Gui Yang

  2. University of Chinese Academy of Sciences, Beijing, China

    Yu-Sheng Chen, Hai-Li Ma, Wei-Yi Lai & Yun-Gui Yang

  3. State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China

    Wei-Yi Lai

Authors
  1. Yu-Sheng Chen
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  2. Hai-Li Ma
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  3. Ying Yang
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  4. Wei-Yi Lai
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  5. Bao-Fa Sun
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  6. Yun-Gui Yang
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Corresponding author

Correspondence to Yun-Gui Yang .

Editor information

Editors and Affiliations

  1. Department of Pathology, Yale University School of Medicine, New Haven, CT, USA

    Narendra Wajapeyee

  2. Yale Cancer Center, Yale University School of Medicine, New Haven, CT, USA

    Romi Gupta

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Chen, YS., Ma, HL., Yang, Y., Lai, WY., Sun, BF., Yang, YG. (2019). 5-Methylcytosine Analysis by RNA-BisSeq. In: Wajapeyee, N., Gupta, R. (eds) Epitranscriptomics. Methods in Molecular Biology, vol 1870. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8808-2_18

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  • DOI: https://doi.org/10.1007/978-1-4939-8808-2_18

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8807-5

  • Online ISBN: 978-1-4939-8808-2

  • eBook Packages: Springer Protocols

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