GSM8860082: R01041RF_Control; Rattus norvegicus; RNA-Seq
Vestibular epithelia were quickly dissected in ice-cold PBS. Two utricles and six crista from each animal were pooled and stored at -80C. Total RNA was extracted using the Qiagen RNeasy mini kit and protocol. RNA sequencing libraries were prepared following the SMARTseq2 protocol (Picelli et al., 2014) with some modifications. Briefly, reverse transcription of the total RNA input material of 1.8 µl (6-11 ng, in function of the sample availability) was performed using SuperScript II (Invitrogen) in the presence of oligo-dT30VN (1µM; 5′-AAG CAG TGG TAT CAA CGC AGA GTA CT30VN-3′), template-switching oligonucleotides (1 µM) and betaine (1M). The cDNA was amplified using the KAPA Hifi Hotstart ReadyMix (2x) (Roche) and 100 nM IS PCR primer (5′-AAG CAG TGG TAT CAA CGC AGA GT-3′) with 8 cycles of PCR amplification. Following purification with Agencourt Ampure XP beads (1:1 ratio; Beckmann Coulter), the product size distribution and the quantity were assessed with a Bioanalyzer High Sensitivity DNA Kit (Agilent). The amplified cDNA (200 ng) was fragmented for 10 min at 55 ºC using Nextera XT (Illumina) and amplified for 12 cycles with indexed Nextera PCR primers. The library was purified twice with Agencourt Ampure XP beads (0.8:1 ratio) and quantified on a Bioanalyzer using a High Sensitivity DNA Kit.