63.3_Male_Cov2_Placebo_5_p
At 21 days post infection, animals were sacrificed; subsequently, the lungs were harvested. Male and female golden hamsters (8-12 weeks old) were purchased from Janvier. All animals were kept under standard housing conditions (21 ± 2 °C, 40–50% humidity, food and water ad libitum) with a 12:12 light–dark cycle at the Leibniz Institute of Virology in Hamburg, Germany. For SARS-CoV-2 infection, hamsters were anaesthetized with 150 mg/kg ketamine and 10 mg kg-1 xylazine by intraperitoneal injection. The animals were intranasally inoculated with 105 plaque forming units (p.f.u.) As control, hamsters were treated by oral gavage with DMSO as vehicle. RNAprotect-fixed tissues were homogenized in 1 ml TRIZOL (Invitrogen) with 5 sterile, stainless steel beads (Ø 2 mm, Retch) at 30 Hz for 10 min in the mixer mill MM400 (Retsch). Cells were lysed in 500 µl or 1 ml TRIZOL (Invitrogen) per well, according to the well size, incubated for 5 min at RT. The cell lysate was resuspended and transferred into a fresh tube. 125 µl or 250 μl chloroform were added respectively to each sample, vortexed for 30 sec and subsequently centrifuged for 15 min at 13,000 g.  Total RNA was isolated from homogenized tissues and the aqueous phase of the lysed cells using the innuPREP RNA Mini Kit 2.0 (Analytik Jena) according to the manufacturer's instructions with an additional on column DNase I treatment using the RNase-free DNase Set (QIAGEN).  Total RNA from formalin-fixed, paraffin-embedded human lung tissue sections was purified using the RNeasy@ FFPE Kit (Qiagen) according to the manufacturer's instructions.  The RNA was eluted in RNase-free water and mixed with 1 U μl-1 RiboLock RNase inhibitor (Thermo Fisher Scientific). Preparation of stranded cDNA libraries was performed with Illumina Stranded mRNA Prep, Ligation kit, starting with 1000 ng total RNA according to the manufacturer's instructions.