Genome-wide profiling of in vivo RNA structure at single-nucleotide resolution using structure-seq

Research output: Journal Publications and Reviews (RGC: 21, 22, 62)21_Publication in refereed journalpeer-review

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Author(s)

  • Yiliang Ding
  • Chun Kit Kwok
  • Yin Tang
  • Philip C. Bevilacqua
  • Sarah M. Assmann

Detail(s)

Original languageEnglish
Pages (from-to)1050-1066
Journal / PublicationNature Protocols
Volume10
Issue number7
Publication statusPublished - 27 Jul 2015
Externally publishedYes

Abstract

Structure-seq is a high-throughput and quantitative method that provides genome-wide information on RNA structure at single-nucleotide resolution. Structure-seq can be performed both in vivo and in vitro to study RNA structure-function relationships, RNA regulation of gene expression and RNA processing. Structure-seq can be carried out by an experienced molecular biologist with a basic understanding of bioinformatics. Structure-seq begins with chemical RNA structure probing under single-hit kinetics conditions. Certain chemical modifications, e.g., methylation of the Watson-Crick face of unpaired adenine and cytosine residues by dimethyl sulfate, result in a stop in reverse transcription. Modified RNA is then subjected to reverse transcription using random hexamer primers, which minimizes 3′ end bias; reverse transcription proceeds until it is blocked by a chemically modified residue. Resultant cDNAs are amplified by adapter-based PCR and subjected to high-throughput sequencing, subsequently allowing retrieval of the structural information on a genome-wide scale. In contrast to classical methods that provide information only on individual transcripts, a single structure-seq experiment provides information on tens of thousands of RNA structures in ∼1 month. Although the procedure described here is for Arabidopsis thaliana seedlings in vivo, structure-seq is widely applicable, thereby opening new avenues to explore RNA structure-function relationships in living organisms.

Citation Format(s)

Genome-wide profiling of in vivo RNA structure at single-nucleotide resolution using structure-seq. / Ding, Yiliang; Kwok, Chun Kit; Tang, Yin; Bevilacqua, Philip C.; Assmann, Sarah M.

In: Nature Protocols, Vol. 10, No. 7, 27.07.2015, p. 1050-1066.

Research output: Journal Publications and Reviews (RGC: 21, 22, 62)21_Publication in refereed journalpeer-review