High-Throughput Mapping of 2′-O-Me Residues in RNA Using Next-Generation Sequencing (Illumina RiboMethSeq Protocol)
| Autor: | Florence Blanloeil-Oillo, Virginie Marchand, Mark Helm, Aseel El Hajj, Lilia Ayadi, Yuri Motorin |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
chemistry.chemical_classification biology Computer science 2'-O-methylation RNA Computational biology 010402 general chemistry biology.organism_classification 01 natural sciences Primer extension DNA sequencing 0104 chemical sciences 03 medical and health sciences 030104 developmental biology chemistry RNA modification DECIPHER Nucleotide Ligation Protocol (object-oriented programming) Throughput (business) Illumina dye sequencing Bacteria Archaea |
| Zdroj: | Methods in Molecular Biology ISBN: 9781493968053 |
| DOI: | 10.1007/978-1-4939-6807-7_12 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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