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Methods Enzymol ; 558: 125-152, 2015.
Article in English | MEDLINE | ID: mdl-26068740

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.


Subject(s)
High-Throughput Screening Assays , Molecular Probes/chemistry , RNA Probes/chemistry , RNA Processing, Post-Transcriptional , RNA, Long Noncoding/chemistry , Software , DNA, Complementary/chemistry , DNA, Complementary/metabolism , Electrophoresis, Polyacrylamide Gel , Nucleic Acid Conformation , Oligonucleotides/chemistry , Oligonucleotides/metabolism , Polymerase Chain Reaction , RNA Folding , RNA, Long Noncoding/metabolism , Reverse Transcription , Small Molecule Libraries/chemistry , Small Molecule Libraries/metabolism
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