GSM5543533: Injured urethra 4A [miRNA-seq]; Rattus norvegicus; miRNA-Seq
Total RNA was extracted from the urethral tissue of the injured and normal rats using the Trizol reagent. Library was prepared with 1 μg total RNA for each sample. Total RNA was purified by electrophoretic separation on a 15% urea denaturing polyacrylamide gel electrophoresis (PAGE) gel and small RNA regions corresponding to the 18–30 nt bands in the marker lane (14-30 ssRNA Ladder Marker, TAKARA) were excised and recovered. Then the 18–30 nt small RNAs were ligated to adenylated 3' adapters annealed to unique molecular identifiers (UMI), followed by the ligation of 5'adapters. The adapter-ligated small RNAs were subsequently transcribed into cDNA by SuperScript II Reverse Transcriptase (Invitrogen, USA) and then several rounds of PCR amplification with PCR Primer Cocktail and PCR Mix were performed to enrich the cDNA fragments. The PCR products were selected by agarose gel electrophoresis with target fragments 110~130 bp, and then purified by QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA). The library was quality and quantitated in two methods: check the distribution of the fragments size using the Agilent 2100 bioanalyzer, and quantify the library using real-time quantitative PCR (QPCR) (TaqMan Probe). The final ligation PCR products were sequenced using the BGISEQ-500 platform (BGI-Shenzhen, China).