Ssoadvance universal supermix

A DNeasy Blood & Tissue Kit (Qiagen) was used to isolate DNA from PMA-treated and non-treated P. carotovorum cultures following the manufacturer’s protocol. Primer set PEC-F (5′-GTGCAAGCGTTAATCGGAATG-3′) and PEC-R (5′-CTCTACAAGACTCTAGCCTGTCAGT TT-3′) targeting the 16S rRNA gene (Pritchard et al., 2013 (link)) was used for SYBR Green qPCR-based specific detection of P. carotovorum; primers were synthesized by Integrated DNA Technologies (IDT, Coralville, IA, United States). SYBR Green qPCR assays were performed in a 25 μl reaction mixture containing 12.5 μl SsoAdvance Universal Supermix (Bio-Rad, Hercules, CA, United States), 8.5 μl of ultrapure water (Thermo Fisher Scientific, Grand Island, NY, United States), and 0.2 μM of each forward and reverse primer. SYBR Green qPCR conditions were 30 s at 95°C followed by 30 cycles of 10 s at 95°C and 30 s at 60°C using CFX96 Real-Time PCR Detection System (Bio-Rad). Three replicates were used for each reaction and water was used as a non-template control. Cycle threshold was set manually, and Bio-Rad CFX Manager 3.1 software was used to analyze the data.

For the exosome-purified pellet, 35 μL of QIAzol was added and proceeded for total RNA isolation including both mRNA and miRNA using miRNeasy Micro Kit (Qiagen, #1071023, Germany) following the provided protocol. For mRNA analysis, weverse transcription was performed to synthesize cDNA using PrimeScript 1st strand cDNA synthesis kit (TAKARA, #6110A, JAPAN). Real-time qPCR for STC2 mRNA was performed with BioRad CFX Connect using SsoAdvance Universal Supermix (BioRad, #1725270, USA). For miRNA analysis, reverse transcription was performed with 10.0 ng RNA using miR-184-specific reverse transcription primer and quantified with miR-184 probe-based real-time qPCR using TaqMan MicroRNA Reverse Transcription Kit (Applied Bioscience, #4366596, US), miR-184-specific TaqMan MicroRNA Assay (Applied Bioscience, #4427975, US), and TaqMan Universal Master Mix II (Applied Bioscience, #4440040, US) following the manufacturer’s protocol. Real-time qPCR was performed with BioRad CFX Connect and analyzed with CFX Maestro software.

Brain samples were homogenized on ice by Dounce homogenizer and total RNA was extracted by TRIzol Reagent (Life Technologies Corporation). Cell samples, total RNA was extracted by RNeasy Mini Kit (QIAGEN) following manufacturer’s protocol. The purity and concentration of RNA were determined by NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific). cDNA first strand was obtained from one microgram of total RNA using iScript™ Reverse Transcription Supermix (Bio-Rad Laboratories, CA, USA). The resulting cDNA was used as template SYBR-green or Eva-green-based real-time PCR quantification using SsoAdvance Universal Supermix (Bio-Rad Laboratories). Data were collected and analyzed using CFX Manager 3.0 software, the ΔΔCt method. Melting curve was produced for every run to assure a unique amplified product per primer set. Primers are depicted in Supplementary Table S4.