GSM9684955: Fibroblast_EV_miRNA_rep2; Rattus norvegicus; miRNA-Seq
Total RNA was extracted from exosomes and intracellular fractions of rat peripheral nerve fibroblasts using a miRNA isolation kit. RNA quantity was determined using NanoDrop 2000, and RNA quality was verified by Agilent 2100 Bioanalyzer. One microgram of qualified total RNA was used for small RNA library preparation. Total RNA was extracted and quantified using the NanoDrop 2000 spectrophotometer. RNA quality was verified using the Agilent 2100 Bioanalyzer. For each sample, 1 μg of total RNA was used for small RNA library preparation. The 3' adapter was first ligated to RNA using T4 RNA Ligase 2 (Deletion Mutant), followed by ligation of the 5' adapter using T4 RNA Ligase. Adapter-ligated RNA was reverse-transcribed into cDNA using SuperScript II Reverse Transcriptase. PCR amplification was then performed with the following conditions: 98°C for 30 s; 15 cycles of 98°C for 10 s, 60°C for 30 s, 72°C for 15 s; and a final extension at 72°C for 10 min. PCR products were separated by gel electrophoresis, and cDNA fragments of 147 nt and 157 nt were excised and purified. Library quality was assessed using the Agilent 2100 Bioanalyzer with a High Sensitivity DNA Chip.