A detailed protocol for stranded RNA-sequencing

RNA-sequencing is a revolutionary tool to follow differential expression after treatment with cancer chemopreventive agents. It allows a real genome-wide screening independent of prior assumptions and is well suited for analyzing coding but also long noncoding RNAs. It still consents the discovery of new genes and isoforms and increased our knowledge of antisense and other noncoding RNAs in a tremendous manner. Moreover, it permits to detect low-abundance and biologically critical isoforms and reveals genetic variants and gene fusions in one single assay.

The development of RNA-sequencing (RNA-Seq) has allowed many advances in the genome-wide transcriptional profiling. RNA-Seq is based on next generation sequencing and consents the quantification of all transcripts longer than 200 nucleotides, including those that are not annotated yet, and therefore still gives rise to the discovery of new genes and transcripts. It revolutionized our understanding of the complexity of transcription. There is no limit of the dynamic range of transcript detection due to the technique, permitting therefore also the detection of rare RNA transcripts and of more differentially expressed genes with a higher fold change. Furthermore, it allows the identification of alternative splicing , allelic-specific expression, and posttranscriptional RNA editing events. But also gene fusions or genetic variants can be reliably detected.

Despite the classical RNA-Seq, there are also several alternative methodologies described in literature. RNA immunoprecipitation sequencing (RIP-Seq) allows mapping RNA-protein interactions. Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Ribo-Seq identifies those mRNAs that are actively translated whereas global run-on sequencing (GRO-Seq) is quantifying transcription by directly measuring nascent RNA production.

To perform a classical RNA-sequencing, there are two decisions to make. The first one involves the rRNA removal that can be done either by poly(A) purification of the RNA or by ribosomal depletion. The latter retains the whole spectrum of RNA transcripts and therefore also transcripts that do not contain a poly(A) tail. The second decision to take is whether the RNA-Seq should be stranded or not. A stranded RNA-Seq allows us to know immediately from which DNA strand a given RNA transcript is deriving from which is helpful for newly discovered transcripts but really indispensable for antisense transcript detection. Additionally it is thought to enhance alignment and transcript annotation.

Here, researchers from the Italian National Cancer Institute describe the protocol for a stranded RNA-sequencing with previous ribosomal depletion, starting from the RNA extraction to the library preparation and the final setting up of the sequencing reaction including all necessary quality control and quantification steps.

Schematic overview of the library preparation steps for stranded RNA-sequencing