GSM7866911: miR137OE #1; Rattus norvegicus; RNA-Seq
Animals were deeply anaesthetised and then perfused with ice-cold Diethyl pyrocarbonate (DEPC) treated Phosphate-buffered saline (PBS). Brains were rapidly dissected and flash-frozen using isopentane. RNA was extracted and isolated from tissue samples using TRIzol Reagent as according to manufacturer's guidelines (Invitrogen, Cat. No. 15596026 and 15596018). mRNA-Seq was conducted using the Illumina (San Diego, CA, USA) Stranded mRNA Prep, Ligation Kit as per the manufacturer's instructions. Briefly, 50 ng of total RNA per sample was subjected to poly(A) enrichment via oligo-dT magnetic beads, followed by heat fragmentation and denaturation (94°C, 8 min). Samples were then reverse transcribed, single adenine nucleotides were ligated to cDNA 3´ ends and pre-index anchors were ligated in preparation for dual indexing. cDNA fragments were subsequently amplified via 14 PCR cycles utilising unique dual i7 and i5 indexes (UDP0XXX). The successful production of ~200-600 bp amplicons was validated using an Agilent (Santa Clara, CA, USA) High Sensitivity DNA Kit and Bioanalyzer instrument, after which samples were pooled in equal concentrations and volumes, denatured and diluted. The final pooled library was subjected to 2x151 cycles of paired end sequencing using a NovaSeq6000 instrument and SP v1.5 flow cell.