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High-Throughput Mapping of 2-O-Me Residues in RNA Using Next-Generation Sequencing (Illumina RiboMethSeq Protocol)

  • Virginie Marchand
  • Lilia Ayadi
  • Aseel El Hajj
  • Florence Blanloeil-Oillo
  • Mark Helm
  • Yuri MotorinEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1562)

Abstract

Detection of RNA modifications in native RNAs is a tedious and laborious task, since the global level of these residues is low and most of the suitable physico-chemical methods require purification of the RNA of interest almost to homogeneity. To overcome these limitations, methods based on RT-driven primer extension have been developed and successfully used, sometimes in combination with a specific chemical treatment. Nowadays, some of these approaches have been coupled to high-throughput sequencing technologies, allowing the access to transcriptome-wide data. RNA 2-O-methylation is one of the ubiquitous nucleotide modifications found in many RNA types from bacteria, archaea, and eukarya. Here, we describe a reliable and optimized protocol based on alkaline fragmentation of total RNA coupled to a commonly used ligation approach followed by Illumina sequencing. We describe the methodology for detection and relative quantification of 2-O-methylations with a high sensitivity and reproducibility even with a limited amount of starting material (1 ng of total RNA). Altogether this technique unlocks a technological barrier since it will be applicable for routine parallel treatment of biological and clinical samples to decipher the functions of 2-O-methylations in pathologies.

Key words

2-O-Methylation High-throughput sequencing RNA modification Ribose methylation Alkaline fragmentation 

Notes

Acknowledgment

This work was supported by joint ANR-DFG grant HTRNAMod (ANR-13-ISV8-0001/HE 3397/8-1) to MH and YM, and AO Lorraine University-Lorraine Region “Aberrant RNA methylation in cancer” funding to YM.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Virginie Marchand
    • 1
    • 2
  • Lilia Ayadi
    • 1
  • Aseel El Hajj
    • 1
  • Florence Blanloeil-Oillo
    • 1
    • 2
  • Mark Helm
    • 3
  • Yuri Motorin
    • 1
    • 2
    Email author
  1. 1.IMoPA UMR7365 CNRS-ULBioPole Lorraine UniversityVandoeuvre-Les-NancyFrance
  2. 2.Next-Generation Sequencing Core Facility, FR3209 BMCT CNRS-ULBioPole Lorraine UniversityVandoeuvre-les-NancyFrance
  3. 3.Institute of Pharmacy and BiochemistryJohannes Gutenberg University MainzMainzGermany

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