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.
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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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Marchand, V., Ayadi, L., El Hajj, A., Blanloeil-Oillo, F., Helm, M., Motorin, Y. (2017). High-Throughput Mapping of 2′-O-Me Residues in RNA Using Next-Generation Sequencing (Illumina RiboMethSeq Protocol). In: Lusser, A. (eds) RNA Methylation. Methods in Molecular Biology, vol 1562. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6807-7_12
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DOI: https://doi.org/10.1007/978-1-4939-6807-7_12
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