title: Mod-seq: A High-Throughput Method for Probing RNA Secondary Structure.
abstract: It has become increasingly clear that large RNA molecules, especially long noncoding RNAs, function in almost all gene regulatory processes (Cech & Steitz, 2014). Many large RNAs appear to be structural scaffolds for assembly of important RNA/protein complexes. However, the structures of most large cellular RNA molecules are currently unknown (Hennelly & Sanbonmatsu, 2012). While chemical probing can reveal single-stranded regions of RNA, traditional approaches to identify sites of chemical modification are time consuming. Mod-seq is a high-throughput method used to map chemical modification sites on RNAs of any size, including complex mixtures of RNA. In this protocol, we describe preparation of Mod-seq high-throughput sequencing libraries from chemically modified RNA. We also describe a software package "Mod-seeker," which is a compilation of scripts written in Python, for the analysis of Mod-seq data. Mod-seeker returns statistically significant modification sites, which can then be used to aid in secondary structure prediction.
authors.affiliation: Department of Biology, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA.
authors.forename: Yizhu
authors.initials: Y
authors.lastname: Lin
authors.affiliation: Department of Biology, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA.
authors.forename: Gemma E
authors.initials: GE
authors.lastname: May
authors.affiliation: Department of Biology, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA. Electronic address: mcmanus@andrew.cmu.edu.
authors.forename: C
authors.initials: C
authors.lastname: Joel McManus
mesh_terms: D018076:DNA, Complementary / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D057166:High-Throughput Screening Assays; D015335:Molecular Probes / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction; D059370:RNA Folding; D015347:RNA Probes / Q000737:chemistry*; D012323:RNA Processing, Post-Transcriptional; D062085:RNA, Long Noncoding / Q000737:chemistry* / Q000378:metabolism; D048348:Reverse Transcription; D054852:Small Molecule Libraries / Q000737:chemistry / Q000378:metabolism; D012984:Software
chemical_list: D018076:DNA, Complementary; D015335:Molecular Probes; D009841:Oligonucleotides; D015347:RNA Probes; D062085:RNA, Long Noncoding; D054852:Small Molecule Libraries
keywords: Chemical probing; RNA; SHAPE; Sequencing; Structure