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

High-throughput RNA sequencing is employed to quantify transcript abundance and for the de novo identification of novel transcripts, including a family of alternatively spliced RNA molecules called circular RNAs (circRNAs). However, quantification of circRNAs between sequencing libraries remains challenging because of RNA complexity in addition to confounding errors introduced during exonuclease digestion, library preparation and sequencing itself. We have developed a set of synthetic circular RNA spike-ins - termed 'SynCRS' - that are directly added to RNA samples before exonuclease digestion and library preparation. These SynCRS are of varying sizes and can be integrated into all NGS workflows and, critically, facilitate the quantitative calibration of circular RNA transcript abundance between samples, tissue types, species and laboratories. Overall design: RNA was harvested from Glioblastoma cell line (U87) and two technical replicates were spiked with 6 SynCRS at varying levels prior to RNase R digestion and RNA sequencing