GSM6214403: sham3; Mus musculus; ncRNA-Seq
Total RNA were isolated using mirVanaTM RNA Isolation Kit according to the manufacturer's instructions. To sequence the circRNA transcriptome, the total RNA of the sample was extracted and the ribosomal RNA was digested with the kit ribo-zero; after that, the linear RNA was removed by RNase R; the enriched circRNA was broken into short fragments by adding interruption reagent, and the interrupted circRNA was used as the template to synthesize one-stranded cDNA with six-base random primers, and then the two-stranded cDNA was prepared. The dUTP was used instead of dTTP in the synthesis of cDNA, and then the different junction was connected, and then one strand containing dUTP was digested by UNG enzyme method, and only the cDNA strand with different junction was retained; the cDNA strand was purified by using the kit; the purified cDNA strand was then end-repaired, A-tail was added and sequencing junction was connected, and then fragment size selection was performed. The purified cDNA strands were then end-repaired, A-tailed and sequenced, followed by fragment size selection, and finally PCR amplification; the constructed libraries were quality-checked by Agilent 2100 Bioanalyzer.