Molecular grade water

RNA was isolated from BMDMs or BMDCs using the RNAqueous-Micro Total RNA Isolation Kit (Invitrogen), and DNAse treated with Turbo DNAse (Invitrogen) according to manufacturer’s instructions. 500ng total RNA was reverse transcribed in 10μL reactions using the iScript cDNA Synthesis Kit (BioRad) according to manufacturer’s instructions and cDNA was diluted 10-fold using molecular grade water (Invitrogen). 2.5μL diluted cDNA was used as template in a 10μL qRT-PCR reaction performed in duplicate using gene-specific primers and Kapa SYBR Green Universal qPCR mix (KAPA Biosystems) according to manufacturer’s instructions using a BioRad CFX Connect Real-Time PCR System. The sequences of gene-specific primers are shown in Table 2. Data was analyzed using Excel and all RNA abundances were calculated by using a standard curve of synthesized template (Integrated DNA Technologies, G-Blocks) and are normalized to ActB (β-actin).

Six hours after N-α-Syn stimulation, BMDCs were harvested, washed, and RNA isolated using the Rneasy mini kit (Qiagen, Germantown, MD) by the manufacturer’s protocol. RNA concentration was determined by UV spectroscopy at 260 nm and 280 nm (ND-100 Nanodrop spectrophotometer, Thermo Scientific, Waltham, MA). Five hundred nanograms of RNA was converted to cDNA using the RevertAID first strand cDNA synthesis kit (Thermo Scientific) following the manufacturer’s protocol. cDNA was added to molecular grade water (Invitrogen, Carlsbad, CA) and 2× RT² SYBR green mastermix (Qiagen), and 25 μl of the mixture was added to each well of a Mouse Inflammatory Response and Autoimmunity array (PAMM-077ZA). PCR was performed in Eppendorf Realplex 2S Mastercycler starting at 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. After 40 cycles, melting curve analysis was performed. The Ct values were determined and the ΔΔCt method was used to determine fold changes relative to cDNA derived from media cultured, unstimulated BMDCs samples using RT² Profiler PCR array data analysis version 3.5.

RNA was extracted using RIBOZOL™ (VWR, Radnor, PA, USA). For larvae, approximately 20 larvae were added to 1 mL RIBOZOL, and for adults, approximately 5 mg of each tissue type (pleopod, hepatopancreas, gill, gut) was pooled and added to 1 mL of RIBOZOL. RNA extraction was carried out following the manufacturer’s protocol, with a final resuspension in 50 μL molecular grade water (Fisher Scientific, Waltham MA, USA). To remove any DNA co-extracted with RNA, a DNAse step was carried out using DNAse1 (Sigma Aldrich, St. Louis, MO, USA), following the manufacturer’s protocol.

R. equi DNA concentration was measured with a Qubit Fluorometer (ThermoFisher, Waltham, MA, US) and Qubit dsDNA BR Assay Kits (ThermoFisher, Waltham, MA, US) and adjusted to 10–15 ng/μL. Ten-fold serial dilutions were made with R. equi extracted DNA in molecular grade water (Fisher Scientific, Pittsburgh, PA, US). The qPCR assay stated above was used to determine the CT values for each dilution. A standard regression curve was constructed with QuantStudio^TM Design and Analysis Software (v 1.5.1, Applied Biosystems, Bedford, MA, US) using linear regression analysis of the log₁₀ sample quantity and the corresponding CT values. Slope and regression equations were calculated for all primer sets individually and in combination. The percentage of efficiency of the qPCR reaction was calculated using the following formula: Efficiency% = (−1 + 10^(−1/slope)) × 100.

Three giant colonies of each culture were selected and transferred to microfuge tubes containing 700 µl of molecular grade water (Fisher). Yeast cells were recovered by centrifugation and DNA was extracted in accordance with the manufacture’s guidelines using a PureLink Microbiome DNA Purification Kit (Invitrogen). Individual strains were identified using the interdelta (ITS) Polymerase chain reaction PCR method^{14 (link),79} ; primers δ2 (5′-GTGGATTTTTATTCCAAC-3′) and δ12 (5′-TCAACAATGGAATCCCAAC-3′), using a BioRad T100 Thermocycler and Invitrogen’s Platinum Hot start PCR Master Mix. PCR products were analysed on an Agilent 2100 Bioanalyzer using the Agilent DNA 7500 chip. The resulting bands were analysed using Minitab 19 Statistical software (2019) hierarchical clustering function with dendrograms produced using Euclidean distance function.

GSK232 was purchased from Cayman Chemical Company (Ann Arbor, MI). A master stock of 1.235 mM GSK232 was prepared by dissolving 5 mg of the compound in 500 μl of high performance liquid chromatography-grade dimethyl sulfoxide packaged under argon gas (Alfa Aesar Co., Ward Hill, Massachusetts). Serial dilutions of GSK232 ranging from 0.1 nM to 4 μM were prepared in molecular-grade water (Fisher Scientific, Fair Lawn, NJ), yielding working stocks with GSK232 concentrations 10-fold greater than the final concentrations used in culture. All stocks of GSK232 were stored at –80°C and were thawed at 37°C immediately before use.

Separate queen body parts and tissues, as well as whole worker bees from the inoculation assays were homogenized using a micro-pestle in a 1.5 mL centrifuge tube, followed by a total RNA extraction with an established TRIzol™ (Invitrogen, Carlsbad, CA, USA) protocol [61 (link)]. The concentration and purity of the extracted RNA samples were measured using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and total RNA concentration was adjusted to 20 ng/µL in molecular grade water (Fisher Scientific, Fair Lawn, NJ, USA). Thereafter, cDNA was synthesized for each sample using the High-Capacity cDNA Reverse-Transcription Kit (Applied Biosystems, Foster City, CA, USA). Ten microliters of the RNA template (20 ng/µL) were added to 10 µL of the provided cDNA master mix, followed by an incubation period as recommended by the manufacturer: 10 min at 25 °C, 120 min at 37 °C, and 5 min at 85 °C. The resulting cDNA solution was then diluted 10-fold in molecular grade water and stored at −80 °C to serve as the template in subsequent qPCR-based analysis of immune gene expression and virus quantification.