Profiling Circular RNAs by RNA-seq

Following extraction, total RNA was treated with RNase R to degrade the linear RNA and purified with RNeasy MinElute Cleanup Kit (Qiagen, Inc., Valencia, CA, USA). Next, a strand-specific library was constructed with VAHTS Total RNA-seq (H/M/R) Library Prep Kit for Illumina according to the manufacturer's protocol. In brief, ribosomal RNA was removed to retain the circRNAs. The enriched circRNAs were broken into short fragments using a fragmentation buffer and reverse transcribed into cDNA with random primers. Secondly, strand cDNA fragments synthesized by DNA polymerase I were purified with VAHTSTM DNA Clean Beads and liquated to Illumina sequencing adapters. Uracil-N-glycosylase was used to digest the second-strand cDNA. The digested products were purified with VAHTSTM DNA Clean Beads, amplified, and sequenced with Illumina HiSeq™ 2500 by Gene Denovo Biotechnology Co. (Guangzhou, China). The edgeR package (http://www.rproject.org/) was used to identify differentially expressed circRNAs. Some significant circRNAs were blasted against the circBase for annotation [13 (link)]. The circRNAs that could not be annotated were defined as novel circRNAs.