GSM5617746: EPS 0h Control rep 4; Mus musculus; RNA-Seq
RNA extraction from C2C12 cells: media was aspirated from each well at the end of an experiment, then cells were immediately lysed in TRI-reagent (Ambion 15599018) with homogenisation achieved via 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 taken for RNA purification with on-column DNase-I treatment, as per manufacturer's directions (Direct-Zol RNA Miniprep #R2052, Zymo Research) followed by additional clean-up step (#R1013, Zymo Research). RNA concentration was confirmed fluorometrically (Qubit, Thermo Fisher) and RNA integrity number (RIN) determined by electrophoresis (Tapestation, Agilent), all RIN were ≥9.5. Stranded libraries were prepared from 500 ng total RNA input with poly-A+ selection (E7760L, NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina®) and included an SIRV Spike-In control, isoform E0 (#025.03, Lexogen) diluted in 1:500. Final library size and concentration were assessed (TapeStation, Agilent). Libraries were pre-sequenced (2 x 150 bp paired-end reads) on the MiniSeq Sequencer (Illumina, San Diego, CA) to obtain the read distribution. Each library was then re-pooled to equal molar concentrations, enzymatically treated, denatured and normalized to 2 nM. Finally, the normalized pooled library was sequenced with 150bp paired end reads on the Illumina NovaSeq 6000 platform (Deakin University Genomics Centre), generating at least 40 million reads per sample.