GSM5957089: r201203; Rattus norvegicus; miRNA-Seq
Total RNA was isolated using the miRvana PARIS kit (AM1556) from Invitrogen according to the manufacturer's protocol. Small-RNA sequencing was performed on three biological replicates per group by Novogene America (Sacramento, CA, USA). Total RNA was assessed for quality before library preparation and RNA Integrity Numbers (RIN) were ≥ 7 with 28S/18S ≥ 1.3. A total amount of 2 μg total RNA per sample was used as input material for the  small RNA library. Sequencing libraries were generated using NEBNext®Multiplex Small RNA Library Prep Set for Illumina® (NEB, USA.) following manufacturer's recommendations. PCR amplification was performed using LongAmp Taq 2X Master Mix, SR Primer for illumina and index (X) primer. PCR products were purified on a 8% polyacrylamide gel (100V, 80 min). DNA  fragments corresponding to 140~160 bp (the length of small noncoding RNA plus the 3' and 5' adaptors) were recovered and dissolved in 8 μL elution buffer. At last, library quality was assessed on the Agilent Bioanalyzer 2100 system using DNA High Sensitivity Chips.