Plant Functional Genomics pp 41-70

Part of the Methods in Molecular Biology book series (MIMB, volume 1284) | Cite as

High-Throughput Nuclease-Mediated Probing of RNA Secondary Structure in Plant Transcriptomes

Protocol

Abstract

Empirical measurement of RNA secondary structure is an invaluable tool that has provided a more complete understanding of the RNA life cycle and functionality of this extremely important molecule. In general, methods for probing structural information involve treating RNA with either a chemical or an enzyme that preferentially targets regions of the RNA in a single- or double-stranded conformation (ssRNA and dsRNA, respectively). Here, we describe an approach that utilizes a combination of ssRNA- and dsRNA-specific nuclease (ss- and dsRNase, respectively) treatments along with high-throughput sequencing technology to provide comprehensive and robust measurements of RNA secondary structure across entire plant transcriptomes.

Key words

RNA genomics RNA secondary structure Posttranscriptional regulation Transcriptome Nuclease probing 

Abbreviations

RBP

RNA binding protein

rRNA

Ribosomal RNA

tRNA

Transfer RNA

snoRNA

Small nucleolar RNA

lncRNA

Long non-coding RNA

ssRNA

Single-stranded RNA

dsRNA

Double-stranded RNA

ssRNase

Single-stranded RNA nuclease

dsRNase

Double-stranded RNA nuclease

PCR

Polymerase chain reaction

bp

Base pair

smRNA

Small RNA

smRNA-seq

Small RNA sequencing

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Lee E. Vandivier
    • 1
    • 2
    • 3
  • Fan Li
    • 1
    • 2
    • 4
  • Brian D. Gregory
    • 1
    • 2
    • 3
    • 4
  1. 1.Department of BiologyUniversity of PennsylvaniaPhiladelphiaUSA
  2. 2.PENN Genome Frontiers InstituteUniversity of PennsylvaniaPhiladelphiaUSA
  3. 3.Cell and Molecular Biology Graduate ProgramUniversity of PennsylvaniaPhiladelphiaUSA
  4. 4.Genomics and Computational Biology Graduate ProgramUniversity of PennsylvaniaPhiladelphiaUSA

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