GSM7817775: DIFF_miR-214-3p_G1; Rattus norvegicus; RNA-Seq
For RNA extraction, TriPure Isolation Reagent (Roche) was added to the washed wells and the plates were stored for minimum 30 minutes at -80°C to help with the cell lysis. RNA was hereafter extracted following the manufacturer's protocol except for the precipitation step, samples were stored for 2h in isopropanol at -20°C to help with RNA precipitation before being centrifuged. For library preparation, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first cDNA strand was synthesized using random hexamer primers, the second using either dUTP for directional library or dTTP for non-directional library, followed by end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion (for the directional library only), amplification, and purification. The libraries, quantified with Qubit and real-time PCR and verified by bioanalyzer for size distribution, were pooled and sequenced on Illumina NovaSeq 6000.