0.45 mm syringe filter

To antagonize miR-21, we used the miRZip-21 lentiviral vector expressing anti-sense miR-21 (Systems Biosciences). miRZip-scrambled hairpin vector was used as a control (System Biosciences). The vector additionally contained a GFP reporter. Lentivirus was produced by transfection of a lentiviral vector, along with psPAX2 (Plasmid #12260; Addgene) and pMD2.G (Plasmid #12259; Add gene) expression vectors into HEK293T cells by using FuGENE (Promega). Lentiviral particles were collected 48 and 72 hours after transfection, filtered through a 0.45-mm syringe filter (Millipore), concentrated using Peg-it solution (System Biosciences) and titered on HEK293T cells. For lentiviral transduction, naive CD4⁺ or CD8⁺ T cells labeled with CellTrace Violet (Thermo Fisher Scientific) were activated with anti-CD3/anti-CD28 beads and transduced with a lentiviral vector expressing scrambled control RNA or anti-sense miR-21 at a multiplicity of infection of 10 in the presence of 8 mg/ml polybrene (Sigma) and 10 U/ml human IL-2 (Peprotech). After 36 hours, activated cells were washed and cultured on plates coated with 1 mg/ml anti-CD3 (CD3–2) plus 2 mg/ml soluble anti-CD28 Ab (CD28.2) and 10 U/ml human IL-2 (Peprotech).

Adult rainbow trout were anesthetized with MS-222, and serum was collected by centrifugation for 15 min at 4 °C, 10,000g. Trout nasal mucus was gently scraped from the olfactory rosette surface, transferred into an Eppendorf tube, vigorously vortexed and centrifuged at 400g for 10 min at 4 °C to remove any trout cells present. To separate the nasal-associated bacteria from the mucus, the cell-free supernatant was thereafter centrifuged at 10,000g for 10 min. The resulting supernatant (nasal mucus free of bacteria) was harvested, filtered with a 0.45-mm syringe filter (Millipore) and stored at −80 °C, whereas the pellet (containing nasal-associated bacteria) was washed three times with PBS (pH 7.2) and resus-pended for further analysis. As positive controls, gut- and skin-associated bacteria and mucus were also collected as explained elsewhere^{5 (link),6 (link)}. In addition, blocks of olfactory tissue (both rosettes) were collected and cryoblocks were made using Optimal Cutting Temperature medium (Tissue-Tek).

The tissue punches of individual mice within an experimental or control group were combined (3 mice/group). Tissue punches of the ventral midbrain were flash frozen in liquid nitrogen for later use or directly homogenized in 2mL of homogenization buffer containing (in mM): 320 Sucrose (sterile filtered), 5 CaCl (sterile filtered), 3 Mg(Ac)2 (sterile filtered), 10 Tris pH 7.8 (sterile filtered), 0.1 EDTA pH 8 (sterile filtered), 0.1% NP40, 0.1 Protease Inhibitor Cocktail (PIC, Sigma), 1 β-mercaptoethanol according to Swiech et al., 2014. Sterile filtration was performed using 0.45 mm syringe filters (Millipore). The volume was then brought up to 5mL using homogenization buffer and was incubated on ice for 5 minutes. For centrifugation, 5mL of 50% Optiprep density gradient medium (Sigma) containing (in mM): 5 CaCl (sterile filtered), 3 Mg(Ac)2 (sterile filtered), 10 Tris pH 7.8 (sterile filtered), 0.1 PIC, 1 β-mercaptoethanol was added to the homogenate and mixed by inversion. The mixture was gently loaded on 10mL of 29% iso-osmolar Optiprep solution in a 1×3 1/2 in Beckman centrifuge tube (SW32 Ti rotor) and spun at 7500 RPM for 30min at 4°C. The cell debris was removed using a KimWipe and the supernatant was gently poured out. The nuclei pellet was vigorously resuspended in sterile 1xPBS and immediately taken to be further processed.