GSM5531343: ID006; Rattus norvegicus; miRNA-Seq
RNA was extracted from rat liver tissue excised at the time of sacrifice and which had been stored at -70°C, using the All Prep DNA/RNA/miRNA Universal Kit (Qiagen, Hilden, Germany) using the manufacturer's instructions. Tissue weight used was ≤ 30 mg, and samples were eluted in 30µl RNase-Free Water. RNA samples were quantified with the Nanodrop 8000 spectrophotometer V2.0 (ThermoScientific, USA) and integrity were checked using the Agilent 2100 Bioanalyser (Agilent Technologies, Waldbronn, Germany). All samples had RNA integrity numbers (RIN) ≥ 7. We created miRNA libraries from 100 ng total RNA using the QIAseq miRNA library kit (Qiagen, Hilden, Germany) according to manufacturer's instructions. This kit was chosen to incorporate unique molecular indices (UMIs) into each cDNA to enable correction for PCR bias. Library concentrations were measured using Qubit™ dsDNA HS Assay Kit (Thermo Fisher, USA). Size of the libraries and calculation of the final molarity for pooling the libraries for sequencing was performed with the High Sensitivity D1000 ScreenTape Assay on a Agilent 2200 TapeStation System (Agilent Technologies, Waldbronn, Germany). Average sizes of the fragments were 181 ± 22 bp, with TapeStation molarity of of 31.8 ± 12.9 nM. Libraries were normalised to 3nM before pooling for sequencing. All samples and all libraries were of good quality and 75 bp single reads were generated for each library using the Illumina NextSeq®500 v2.5 High-output 150 cycle kit (Illumina Inc., Cambridge, UK). A total of 111,746,220 reads were generated for these 24 samples (4,656,093 ± 2,282,243 reads per sample).