title: 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.
authors.affiliation: IMoPA UMR7365 CNRS-UL, BioPole Lorraine University, 9 Avenue de la Foret de Haye, 54505, Vandoeuvre-Les-Nancy, France.|Next-Generation Sequencing Core Facility, FR3209 BMCT CNRS-UL, BioPole Lorraine University, 9 Avenue de la Foret de Haye, 54505, Vandoeuvre-les-Nancy, France.
authors.forename: Virginie
authors.initials: V
authors.lastname: Marchand
authors.affiliation: IMoPA UMR7365 CNRS-UL, BioPole Lorraine University, 9 Avenue de la Foret de Haye, 54505, Vandoeuvre-Les-Nancy, France.
authors.forename: Lilia
authors.initials: L
authors.lastname: Ayadi
authors.affiliation: IMoPA UMR7365 CNRS-UL, BioPole Lorraine University, 9 Avenue de la Foret de Haye, 54505, Vandoeuvre-Les-Nancy, France.
authors.forename: Aseel
authors.initials: A
authors.lastname: El Hajj
authors.affiliation: IMoPA UMR7365 CNRS-UL, BioPole Lorraine University, 9 Avenue de la Foret de Haye, 54505, Vandoeuvre-Les-Nancy, France.|Next-Generation Sequencing Core Facility, FR3209 BMCT CNRS-UL, BioPole Lorraine University, 9 Avenue de la Foret de Haye, 54505, Vandoeuvre-les-Nancy, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Blanloeil-Oillo
authors.affiliation: Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, Staudingerweg 5, 55128, Mainz, Germany.
authors.forename: Mark
authors.initials: M
authors.lastname: Helm
authors.affiliation: IMoPA UMR7365 CNRS-UL, BioPole Lorraine University, 9 Avenue de la Foret de Haye, 54505, Vandoeuvre-Les-Nancy, France. Yuri.Motorin@univ-lorraine.fr.|Next-Generation Sequencing Core Facility, FR3209 BMCT CNRS-UL, BioPole Lorraine University, 9 Avenue de la Foret de Haye, 54505, Vandoeuvre-les-Nancy, France. Yuri.Motorin@univ-lorraine.fr.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Motorin
mesh_terms: D019295:Computational Biology / Q000379:methods; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006868:Hydrolysis; D008745:Methylation; D011786:Quality Control; D012313:RNA / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D017423:Sequence Analysis, RNA / Q000379:methods; D012984:Software
chemical_list: D012313:RNA
keywords: 2′-O-Methylation; Alkaline fragmentation; High-throughput sequencing; RNA modification; Ribose methylation