NextSeq 500 sequencing; COVID-19-associated olfactory dysfunction reveals SARS-CoV-2 neuroinvasion and persistence in the olfactory system
At predefined time-points post-infection, animals were submitted to behavioral tests or euthanized, when samples of nasal turbinates, trachea, lungs and the brain (separated in olfactory bulbs, cerebellum, cortex and brainstem) were collected, immediately frozen at -80°C or formalin-fixed after transcardial perfusion with a physiological solution containing 5.103 U/mL heparin (choay, Sanofi) followed by 4% neutral buffered formaldehyde. Frozen tissues were homogenized with Trizol (15596026, Invitrogen) in Lysing Matrix D 2 mL tubes (116913100, MP Biomedicals) using the FastPrep-24™ system (MP Biomedicals) at the speed of 6.5 m/s during 1 min. Total RNA was extracted using the Direct-zol RNA MicroPrep Kit (R2062, Zymo Research). RNA preparation was used to construct strand specific single end cDNA libraries according to manufacturers' instructions (Truseq Stranded mRNA sample prep kit, Illumina)