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

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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https://hal.univ-lorraine.fr/hal-01799289
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Submitted on : Friday, March 11, 2022 - 5:47:34 PM
Last modification on : Thursday, May 19, 2022 - 1:52:03 PM

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Virginie Marchand, Lilia Ayadi, Aseel El Hajj, Florence Blanloeil-Oillo, Mark Helm, et al.. High-Throughput Mapping of 2'-O-Me Residues in RNA Using Next-Generation Sequencing (Illumina RiboMethSeq Protocol). Methods in Molecular Biology, Humana Press/Springer Imprint, 2017, 1562, pp.171-187. ⟨10.1007/978-1-4939-6807-7_12⟩. ⟨hal-01799289⟩

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