Abstract
RNA molecules play important roles in almost every cellular process, and their functions are mediated by their sequence and structure. Determining the secondary structure of RNAs is central to understanding RNA function and evolution. RNA structure probing techniques coupled to high-throughput sequencing allow determining structural features of RNA molecules at transcriptome-wide scales. Our group recently developed a novel Illumina-based implementation of in vitro parallel probing of RNA structures called nextPARS.Here, we describe a protocol for the computation of the nextPARS scores and their use to obtain the structural profile (single- or double-stranded state) of an RNA sequence at single-nucleotide resolution.
Highlights
Knowledge of the secondary structure of RNAs both in vivo and in vitro is crucial for understanding the regulatory roles that RNAs exert in most cell functions, via characterizing their intramolecular interactions, and how they can change depending on external conditions, including interactions with other molecules [1]
Among several approaches that have been described during the last years to interrogate RNA structure using high throughput sequencing technologies, nextPARS [2] is an enzymatic-based technique that allows probing the secondary structure of RNAs in vitro at a genome-wide scale
It is an adaptation of the previously developed PARS [3] strategy to the Illumina sequencing platform, which allows for sample multiplexing and higher throughput than the original technique
Summary
Knowledge of the secondary structure of RNAs both in vivo and in vitro is crucial for understanding the regulatory roles that RNAs exert in most cell functions, via characterizing their intramolecular interactions, and how they can change depending on external conditions, including interactions with other molecules [1]. Among several approaches that have been described during the last years to interrogate RNA structure using high throughput sequencing technologies, nextPARS [2] is an enzymatic-based technique that allows probing the secondary structure of RNAs in vitro at a genome-wide scale. It is an adaptation of the previously developed PARS [3] strategy to the Illumina sequencing platform, which allows for sample multiplexing and higher throughput than the original technique. We briefly describe nextPARS methodology and report, using an illustrative example, how the nextPARS raw output data is analyzed in order to go from sequencing reads to structural profiles of RNAs present in a sample (Fig. 1)
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