Mini beadbeater 96 cell disrupter

To perform initial exploratory metagenomic analysis, we selected 20 S. murinus and pooled their tissues for RNA extraction and sequencing. Approximately 1–2 g of tissue (lung, spleen, and kidney) was homogenized in 1 ml of AVL buffer (Qiagen, Hilden, Germany) using silicon carbide shards and a Mini-Beadbeater-96 cell disrupter (Biospec Products, Bartlesville, USA) for 1 min at 2000 rpm. The homogenates were pooled and processed following our previous protocol [23 (link)], and RNA was extracted using Direct-zol RNA miniprep kit (Zymo Research Corporation, Irvine, USA) as per the manufacturer's instructions. For S. murinus (n = 37) molecular screening and subsequent sequencing, the lung, spleen, and kidney tissues from each individual shrew were separately weighed (1.3 to 27.6 mg) and homogenized in 500 μl of AVL buffer (Qiagen, Hilden, Germany). The whole tick was homogenized as above. Homogenates were centrifuged at 13,000 rpm for 1 min, and the RNA extraction was performed on the supernatant as described above.

At experimental endpoint, mice were euthanized by intraperitoneal injection of sodium pentobarbital (120 mg/kg), followed by cardiac puncture for blood collection, nasal lavage, and excision of lungs and heads. For nasal lavage, an incision was made one-third of the way down the trachea and one ml of PBS was rinsed through the sinus cavity using an intravenous catheter. For lung tissues, whole lungs were collected in sterile PBS, and homogenized using a Mini Beadbeater-96 cell disrupter (BioSpec Products, Oklahoma, USA) for 5 min. Serial dilutions of nasal lavage fluid and lung homogenate were plated on LB agar for bacterial enumeration, and the remaining liquid was stored at -20°C for use in protein quantification and ELISA.
For histopathological studies of the nasal cavity, mice were decapitated and the head was degloved for optimal perfusion of fixative (10% buffered formalin) into the tissue. Decalcification, cross-sectioning and histochemical staining with hematoxylin and eosin (H&E), of samples were adapted from Lindsay et al. (2006) (link) and performed by Wax-It Histology Services Inc. (University of British Columbia, Vancouver, CA) or ourselves. Histological evaluation was performed on samples post-infection, and representative images are shown.