GSM9726619: MW_2_circRNA; Rattus norvegicus; ncRNA-Seq
Total RNA was extracted from tissue samples using TRIzol reagent following the manufacturer's protocol. To enrich circular RNAs, linear RNAs were digested with RNase R (3 U/μg RNA, 37°C for 30 minutes). The treated RNA was purified by phenol-chloroform extraction and ethanol precipitation. RNA integrity and concentration were assessed using a Bioanalyzer 2100. Purified circRNA was stored at -80°C before library preparation. Enriched circRNA was fragmented to 150–300 nt, then reverse transcribed using random hexamers. Second-strand synthesis generated double-stranded cDNA, followed by end repair, A-tailing, and adapter ligation. Libraries were amplified by 12–15 PCR cycles, size-selected (300–400 bp) on agarose gels, and quantified by Qubit. Sequencing was performed on Illumina NovaSeq 6000 (150 bp paired-end).