Protocol

Yeast Functional Genomics

Volume 1361 of the series Methods in Molecular Biology pp 141-160

Genome-Wide Probing of RNA Structures In Vitro Using Nucleases and Deep Sequencing

  • Yue WanAffiliated withStem Cell and Developmental Biology, Genome Institute of Singapore Email author 
  • , Kun QuAffiliated withHoward Hughes Medical Institute and Program in Epithelial Biology, Stanford University School of Medicine
  • , Zhengqing OuyangAffiliated withThe Jackson Laboratory for Genomic MedicineDepartment of Biomedical Engineering, University of Connecticut
  • , Howard Y. ChangAffiliated withHoward Hughes Medical Institute and Program in Epithelial Biology, Stanford University School of Medicine

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Abstract

RNA structure probing is an important technique that studies the secondary and tertiary conformations of an RNA. While it was traditionally performed on one RNA at a time, recent advances in deep sequencing has enabled the secondary structure mapping of thousands of RNAs simultaneously. Here, we describe the method Parallel Analysis for RNA Structures (PARS), which couples double and single strand specific nuclease probing to high throughput sequencing. Upon cloning of the cleavage sites into a cDNA library, deep sequencing and mapping of reads to the transcriptome, the position of paired and unpaired bases along cellular RNAs can be identified. PARS can be performed under diverse solution conditions and on different organismal RNAs to provide genome-wide RNA structural information. This information can also be further used to constrain computational predictions to provide better RNA structure models under different conditions.

Key words

RNA Structure Biochemistry Genomics High-throughput sequencing