Use of synthetic circular RNA spike-ins (SynCRS) for normalization of circular RNA sequencing data

High-throughput RNA sequencing enables the quantification of transcript abundance and the identification of novel transcripts in biological samples. These include circular RNAs (circRNAs), a family of alternatively spliced RNA molecules that form a continuous loop. However, quantification and comparison of circRNAs between RNA sequencing libraries remain challenging due to confounding errors introduced during exonuclease digestion, library preparation and RNA sequencing itself. Here we describe a set of synthetic circRNA spike-ins-termed 'SynCRS'-that can be added directly to purified RNA samples before exonuclease digestion and library preparation. SynCRS, introduced either individually or in combinations of varying size and abundance, can be integrated into all next-generation sequencing workflows and, critically, facilitate the quantitative calibration of circRNA transcript abundance between samples, tissue types, species and laboratories. Our step-by-step protocol details the generation of SynCRS and guides users on the stoichiometry of SynCRS spike-in to RNA samples, followed by the bioinformatic steps required to facilitate quantitative comparisons of circRNAs between libraries. The laboratory steps to produce the SynCRS require an additional 3 d on top of the high throughput circRNA sequencing and bioinformatics. The protocol is suitable for users with basic experience in molecular biology and bioinformatics. Synthetic circular RNA spike-ins facilitate the normalization of circular RNA transcript abundance across two or more RNA sequencing datasets.The procedure covers preparation of synthetic circular RNA spike-ins, spike-in guidelines and the bioinformatic steps required for their quantification to facilitate comparisons between libraries. This protocol shows distinct advantages over existing strategies for normalizing circular RNA abundance in RNA sequencing libraries. This protocol presents a method for calibrating circular RNA abundance for comparison between RNA sequencing libraries, utilizing a spike-in of synthetic circular RNAs.