title: GSM6965024: Mitochondrial ribosome IP_Vehicle_Rep3 [IPm_V3]; Mus musculus; RNA-Seq
library_construction_protocol: C57Bl/6 mice were euthanized by asphyxiation with CO2 two hours post-treatment (rapamycin or vehicle). Then, vestibular nuclei were dissected according to the Paxinos mouse brain atlas using a brain matrix. Brain slices were immediately frozen by liquid nitrogen and conserved at -80ªC until processing. A pool of four vestibular nuclei was dissociated with an ice-cold Dounce homogenizer containing a defined homogenization buffer. Subsequently, a double immunocapture method was used to capture cytosolic and mitochondrial ribosomes from vestibular glutamatergic neurons while preserving RNA integrity. Finally, RNeasy Micro kit (Qiagen) was applied to extract RNA according manufacturer's instructions. RNA quantification was assessed by Quant-iT RiboGreen RNA Reagent kit and RIN was analyzed on an Agilent 2100 Bioanalyzer system with the RNA 6000 Nano and Pico kits. Samples were conserved at -80ºC until sequencing library preparation. RNA libraries for RNA-seq were prepared using TrioRNASeq Nugen (AnyDeplete Mouse) Kit following manufacturer's protocols (starting input: 6ng, Number of PCR amplification cycles: 5x).