Minibeater 16

Colon samples (2–10 mg) were thawed in cold water and weighed (XS105 DualRange, Mettler Toledo, Columbus, OH) (2–10 mg). Ice-cold solvent, 2:5:2 CDCl₃/CD₃OH/H₂O (v/v/v), was added (850 μL) with zirconia beads (0.05 mm, BioSpec Products, Bartlesville, OK). Deuterated solvents were used to enable LC-MS and NMR analysis. Samples were ground (Minibeater-16, BioSpec Products, Bartlesville, OK) for 3 min, cooled on ice for 5 min, repeated for 3 cycles. Ground samples were centrifuged for 2 min at 12,000 × g at 4 °C (Microfuge 22R, Beckman Coulter, Brea, CA), the supernatant (750 μL) was centrifuged a second time to remove particulate. Extracts were stored at −80 °C.

Pooled organs (liver, kidney, heart, and spleen) and separate brain samples were collected from each largemouth bass in the immersion experiment (described above) and samples were homogenized (Minibeater-16, BioSpec Products) with 500 μL HMEM (Minimal Essential Medium with Hank’s balanced salts solution) and a 1.3 mm chrome bead for 60 s. Homogenate was spun (centrifuged for 2 min at 8000 rpm) and supernatant was used for RNA extraction (Life Technologies Viral Isolation Kit and KingFisher MagMax instrument). Total RNA was quantified using a NanoVue spectrophotometer (GE Healthcare). The presence of VHSV was measured with a method of RT-qPCR assay as previously described (Hope et al., 2010 (link)) and modified (Cornwell et al., 2012 ).