Circular RNA Profiling and Validation

Random primers and Reverse Transcription Kit (#RR037A, Takara) were used to obtain cDNA according to the manufacturer’s protocol. CircPrimer 2.0 software was used to annotate and obtain circRNA sequences (Zhong et al., 2018 ). Then, the divergent primers, which coved the back-splicing regions, were designed by Primer3¹ (Untergasser et al., 2012 (link)). The PCR products of divergent primers were sequenced to validate the corresponding back-splicing sites. The relative expression levels of selected circRNAs were detected by qRT-PCR using TB Green Premix Ex II (#RR820A, Takara) on a LightCycler 96 system (Roche, Germany) according to the instructions. 18S was used to normalize the threshold cycle (Ct) values, and gene expression was quantified using the relative quantitation method (2^–ΔΔCt). All experimental data are presented as means ± SD.