GSM7653328: H9c2 cells, control 1; Rattus norvegicus; RNA-Seq
Media was aspirated then cells were immediately lysed in 900 µL TRI-reagent (#15599018 Ambion) with homogenisation achieved by pipette mixing. Lysates were then frozen at -80℃ until RNA isolation. TRI-reagent lysates were thawed on ice and centrifuged at 10,000 x g for 10 min at 4℃. An aliquot of the supernatant was transferred to a new tube and mixed with an equal volume of 96% ethanol, then transferred to a spin column for RNA purification with on-column DNase-I treatment, as per manufacturer's directions (#R2052 Direct-Zol RNA Miniprep, Zymo Research). RNA concentration and purity (260:280 nm: >2.0) was determined with a spectrophotometer (NanoDrop 1000, Thermo Fisher Scientific). Library construction was conducted by Macrogen Oceania. RNA concentration was confirmed fluorometrically (Qubit, Thermo Fisher) and RNA integrity number (≥9) was determined by electrophoresis (Tapestation, Agilent). Stranded libraries were prepared from 500 ng total RNA input with poly-A+ selection (TruSeq Stranded mRNA, Illumina). Final library size and concentration were assessed (Qubit, Thermo Fisher and TapeStation, Agilent).