title: RNA-seq of Mesocricetus auratus brainstems during Sars-CoV-2 infection (4 days post infection)
design: RNA-seq of Mesocricetus auratus brainstems during Sars-CoV-2 infection (4 days post infection)
library_construction_protocol: At 4-days post-infection, the animals were euthanized with an excess of anesthetics (ketamine and xylazine) and exsanguination 74. The brain was extracted from the skull, the two brain hemispheres were separated by a median incision and macroscopically divided in four regions using tweezers: (1) olfactory bulbs, (2) cerebellum, (3) cerebral cortex (containing the cortex, the striatum and the hippocampus) and (4) brainstem (containing the diencephalon, the midbrain, the pons and the medulla oblongata) (Figure 2). The lungs were extracted from the thorax and weighted. The nasal turbinates were extracted by opening of the nasal cavity after incision of the nasal and frontal bones. The samples were immediately frozen at -80°C. Frozen samples were weighed and transferred to Lysing Matrix M 2mL tubes (116923050-CF, MP Biomedicals) containing 1mL of ice-cold DMEM (Dulbecco's Modified Eagle medium, Gibco) supplemented with 1% penicillin/streptomycin (15140148, Thermo Fisher). Samples were homogenized using the FastPrep-24™ system (MP Biomedicals), and the following scheme: homogenization at 4.0 m/s for 20 sec, incubation at 4°C for 2 min, and further homogenization at 4.0 m/s for 20 sec. The tubes were centrifuged at 10,000 × g during 2 min at 4 °C, and the supernatants collected and stored at -80°C until further analysis.  Total RNA from brainstem was extracted using the Direct-zol RNA MiniPrep kit (R2052, Zymo Research). Total RNA from nasal turbinates and olfactory bulbs was extracted using the Direct-zol RNA MicroPrep kit (R2062, Zymo Research). In both cases, 125µL of tissue homogenate were incubated with 375µL of Trizol LS (10296028, Invitrogen) and the extraction was performed according to the manufacturer's instructions. RNA preparation was used to build libraries using a Illumina Stranded mRNA library Preparation Kit (Illumina, USA) following the manufacturer's protocol. Quality control was performed on an Agilent BioAnalyzer.