Circular RNA Profiling in Rat Bladder

Total RNA samples were extracted from rat bladder tissues using the Eastep Super Total RNA Extraction Kit (#LS1040; Promega) according to the manufacturer’s instructions. The profiling and quantitation of differentially expressed circular RNAs in rat bladder tissues were done via the RNA sequencing assay as previously described with minor modifications (Fu et al., 2017 (link)). Briefly, total RNA samples were incubated with DNase I (Promega), followed by removal of rRNA components using the Ribo-off rRNA Depletion Kit (#N406-01; Vazyme, Nanjing, and China) following the manufacturer’s instructions. Linear RNA molecules in total RNA samples were then deleted by treating with the RNase R. The resultant circular RNAs were fragmented and subjected to construction of RNA sequencing library via random priming using the Ultr II RNA Library Preparation Kit (#E7770S; NEB, United States) as instructed by the producer. Following end repair, dA-tailing, ligation of adaptors and uracil-specific excision, and the library was finally enriched by PCR method. After size selection, the library was then sequenced with an Illumina Hiseq X ten sequencing system.