Precellys evolution tissue homogenizer

Nasal wash samples were 10-fold serially diluted and added to a 24-well plate containing freshly confluent with Vero E6 cells. For tissue samples, entire trachea or nasal turbinates or the left lobe of the lung (~0.2 g) were resuspended in 1 milliliter MEM and homogenized on a Precellys Evolution tissue homogenizer with a Cooling Unit (Bertin). Tissue homogenates were then 10-fold serially diluted and added to Vero E6. After 1 h the mixture was removed and replenished with Tragacanth gum overlay (final concentration 0.3%). Cells were incubated at 37 °C and 5% CO₂ for 2 days, then fixed with 4% paraformaldehyde, followed by staining of cells with 0.1% crystal violet in 20% methanol for 5–10 min. The infectious titers were then calculated and plotted as plaque forming units per milliliter (PFU/ml).

A total of 90 honey bees (30 winter bees, 30 newly emerged bees, and 30 foragers with pollen) from each Apis mellifera ligustica colony were sampled and stored at −80 °C. Three honey bees from each colony were analyzed. They were added to CKmix50_2 mL tubes (Precellys Lysing Kit) containing ceramic beads (MP Biochemicals GmbH, Eschwege, Germany). Cold RLD buffer (1 mL) was added to the tubes, and they were homogenized using a Precellys Evolution Tissue Homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France) following the manufacturer’s recommended program. A total of 600 µL supernatant with debris was transferred to 1.5 mL Eppendorf tubes, and 400 µL of cold RLD buffer was added. The subsequent steps of RNA isolation followed the Quick-Start Protocol (Clear-STM Total RNA Extraction Kit, in VIRUStech, Republic of Korea). The final volume was 30 µL of total RNA in Elution Buffer. The total RNA concentration and purity were quantified using OD260/OD280 values between 1.8 and 2.0. Next, reverse transcription was performed using the RNA to cDNA EcoDryTM Premix (Oligo dT) kit (Takara, San Jose, CA, USA). Each reverse transcription reaction mixture included 50 ng/µL of total RNA (with the clear volume calculated for each sample) and RNase-free water for a total volume of 20 µL. Reverse transcription was conducted at 42 °C for 60 min, followed by heating at 70 °C for 10 min.

Total RNA was isolated from brain organoids with RNeasy Plus Universal Tissue Mini kit (Qiagen) and Precellys Evolution tissue homogenizer (Bertin). The concentration and purity of RNA samples were checked using the NanoDrop ND-1000 spectrophotometer at 260 and 280  nm (NanoDrop Technologies); the A260/280 ratio ranged from 1.8 to 2.2. One μg of total RNA was converted to cDNA using random primers in a volume of 20  μl using an RT2 HT first stand kit (Qiagen) according to the manufacturer’s protocol. cDNA was diluted with sterile water to a final volume of 200  μL. Quantitative expression of markers were determined using 2  µl of diluted cDNA for each primer (Supplementary file 8) set at 10  µM using iQ SYBR Green Supermix (Biorad) in a final volume of 12  µl. Thermocycling was carried out in the CFX96 RT-PCR detection system (Bio-Rad) using SYBR green fluorescence detection. The amplification cycle used was as follows: 10  min at 95  °C, followed by 40 amplification cycles at 95  °C for 15  s, 60  °C for 60  s, and 72  °C for 30  s to reinitialize the cycle again. The specificity of each reaction was also assessed by melting curve analysis to ensure primer specificity. Relative gene expression values were calculated as 2^−ΔCT, where ΔCT is the difference between the cycle threshold (CT) values for genes of interest and housekeeping gene (GAPDH).