Bead mill 24

Brains analyzed in this work were prepared as previously described with the numbering of patients following that in Table 1 in [21 (link)]. Briefly, one half of a patient brain was formalin fixed and the other half frozen. Diagnoses of fixed tissues were made using the stains described in [6 (link), 21 (link)]. Tissue samples in this study were collected from frontal cortex except for CTE and primary age-related tauopathy (PART) samples which were from temporal cortex. 10% w/v brain homogenates of frontal cortex tissue were prepared in ice-cold PBS using 1 mm silica beads (BioSpec, 11079110z) and Beadbeater (BioSpec) or BeadMill 24 (Fischer). K. N. provided CTE; B. G. provided all other samples, PiD, AD, diseases with 4R tau deposits, and controls lacking tau pathology.

To evaluate viral infection, VSV N gene expression in the lungs was measured by quantitative polymerase chain reaction^{9 (link)}. The lungs were homogenized in a tube containing glass beads and 500 μL of pre-chilled phosphate-buffered saline using BeadMill 24 (Thermo Fisher Scientific, MA) at a speed of 6 m/s for 60 s. Each 20 μL of homogenized lung solution was immediately mixed with 500 μL of Isogen (Nippon Gene, Toyama, Japan) and stored at −80 °C until subsequent use. Total RNA was extracted using a QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Total RNA was reverse-transcribed into cDNA and then amplified using Thunderbird SYBR qPCR/RT Set III (TOYOBO Co.., Tsuruga, Japan). VSV N expression was normalized to that of γ-actin^{10 (link)}.

Total RNA from a pool of 2 randomly chosen male and female flies fed on each host from each temporal replicate (n = 3 pools of 2 adult flies) were isolated separately with a Tri-reagent (NZYol, NZYTech, Lisbon, Portugal) following the manufacturers’ instructions for small amounts of tissue. Flies were homogenized in 800 µL of NZYol in a FischerBrand™ BeadMill 24 with one 2.8 mm ceramic bead at 2.4 m/s for 60 s (ThermoFischer Scientific, Massachusetts, USA). The aqueous phase was transferred to a new microtube and, after adding chloroform, samples were centrifuged at 12,000×g for 15 min in a pre-chilled (4 °C) centrifuge (Micro Star 17R, VWR, Pennsylvania, USA). RNA was precipitated with isopropanol for 1 h at − 20 °C. After washing with EtOH 70%, the pellet was air-dried, and then resuspended in 80 µL of DEPC-treated H₂O. DNAse treatment was applied to all samples with DNAse I according to the manufacturers’ instructions (NZYTech, Lisbon, Portugal). RNA was stored at − 80 °C until use.

C. briggsae prg-1 mutant and its wild-type siblings were grown continuously. Approximately 100,000 synchronized L1 animals at ~10^th generation were obtained and plated on 135 mm NGM plates seeded with OP50 and grown at 20°C until the animals reached gravid adult stage. Gravid adult populations were harvested using M9, and subsequently suspended in Trizol. Worms were lysed using the Bead Mill 24 homogenizer (Thermo Fisher Scientific). Bromochloropropane was added to the lysis to perform RNA extraction. Isopropanol was then used to precipitate RNA from the aqueous phase. Small RNAs were isolated from total RNAs using the miRVana miRNA isolation kit (Thermo Fisher Scientific) according to the manufacturer’s protocol.

The right lungs were soaked in Buffer RLT (Qiagen, Tokyo, Japan) and homogenized using a Bead Mill 24 (Thermo Fisher Scientific, Waltham, Tokyo, Japan). Homogenized samples were centrifuged at 12,000 rpm for 3 min, and total RNA was extracted using an RNeasy mini kit (Qiagen, Tokyo, Japan). RNA concentrations were evaluated using a NanoDrop (Thermo Fisher Scientific, Tokyo, Japan). Complementary DNA was prepared using a Ready-to-Go-T-primed first-strand kit (GE Healthcare, Little Chalfont, UK). Quantitative real-time PCR was performed using an Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific, Tokyo, Japan). The PCR mixture contained 1 µL of cDNA samples and primers of each of the target genes. TaqMan gene expression assays (Thermo Fisher Scientific, Tokyo, Japan) for CXCL1 (Mm04207460_m1), CXCL2 (Mm00436450_m1), TNF-alpha (Mm00443258_m1), G-CSF (Mm00432735_m1), Nrf2 (Mm_00477784_m1), and 36B4 (Mm00725448_s1) were performed. We used 36B4 as an internal control gene and the relative levels of each mRNA were normalized to 36B4 mRNA levels using the ΔΔCT method.

Leaf tissue from each sample was placed into sterile 2-ml conical screw-cap microcentrifuge tubes (Fisherbrand, Pittsburgh, PA, United States) with sterile 13-mm zirconia/silica beads (BioSpec Products, OK, United States), and submerged into liquid nitrogen for 2 min. Samples were homogenized using a Bead Mill 24 (Thermo Fisher Scientific, Walther, MA, United States) for 20 s twice with 5 min in liquid nitrogen between the homogenization steps. DNA extraction was completed using a modified protocol of the DNeasy Plant Mini kit (Qiagen, Valencia, CA, United States) by including 2% v/v liquid polyvinylpyrrolidone and 4 μl of RNase A in the lysis buffer. After addition of P3 buffer, samples were frozen (−20°C) for 1 h. Genomic DNA was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, United States) and stored at −20°C.

Ticks were washed in ethanol and distilled water and then morphologically determined to the species level using standard keys [37 (link),38 (link)]. Prior to extraction, tissues and ticks were crushed in microtubes filled with six metal beads using a fisher brand Bead Mill 24 (Thermofisher, Waltham, MA, USA) at 5500 rpm for 20 s. DNA from ticks and tissues was extracted using a Nucleospin tissue kit (Macherey-Nagel, Düren, Germany). For the blood samples, a Nucleospin Quickpure Blood kit (Macherey-Nagel, Düren, Germany) was used, according to the manufacturer’s instructions. All the ticks were analyzed individually.
To improve the detection of pathogen DNA, total DNA was pre-amplified using a PreAmp Master Mix, according to the manufacturer’s instructions (Fluidigm, South San Francisco, CA, USA). Prior to experiments, primers targeting all pathogens were pooled in equal volumes (200 nM each). The experiment was performed with 1 µL of PreAmp Master Mix, 1.25 µL of pooled primers mix, 1.5 µL distilled water, and 1.25 µL DNA for 5 µL final volume. The pre-amplifications were performed with one cycle at 95 °C for 2 min, 14 cycles at 95 °C for 15 s and 60 °C for 4 min. At the end of pre-amplification, samples were diluted to 1:10. Pre-amplified DNA was stored at −20 °C until further use.