Rnazol rt

RNA was extracted using either the RNA extraction buffers TRIzol (Invitrogen), RNA Bee (Amsbio), and RNAzol RT (Sigma) or an Isolate II RNA minikit (Bioline) according to the manufacturers’ instructions. RT-PCR was performed as described previously (68 (link)). Each primer pair was designed with at least one of them spanning the exon-intron boundary so that mRNA but not genomic DNA was amplified (Table 1) (66 (link), 69 (link)). The cycling parameters were initial denaturation at 94°C for 2 min followed by 25 to 40 cycles of denaturation at 94°C for 10s, annealing at 50 to 55°C for 30 s, and elongation at 68°C for 45 s with a final extension at 68°C for 7 min. PCR band intensity was measured using ImageJ (NIH) or ImageLab 6.0.1 (Bio-Rad) and normalized against an internal control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Low-exposure gels were used in image quantification.
Real-time RT-qPCR was performed using 1/50 of the RT reaction mixture in Power SYBR green PCR master mix (Applied Biosystems) and 300 nM sense and anti-sense primers (Table 1). RT-qPCR was run on a StepOnePlus system (Applied Biosystems) using 10 s denaturation at 95°C followed by 40 cycles of 95°C for 15 s and 60°C for 60 s; melting curves were generated using 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s. Data were analyzed by the 2^−ΔΔCT method using GAPDH as an endogenous control.

Fungal strains were cultivated in Erlenmeyer flasks for 48 h, mycelia and supernatants were separated by filtration through Miracloth (Merck Millipore, part of Merck KGaA, Darmstadt, Germany). Approx. 0.05 g of harvested mycelia were resuspended in 1 ml RNAzol RT (Sigma-Aldrich) and lyzed using a Fast-Prep-24 (MP Biomedicals, Santa Ana, CA, USA) with 0.37 g of small glass beads (0.1 mm diameter), 0.24 g of medium glass beads (1 mm diameter), and a single large glass bead (5 mm diameter) at 6 m/s for 30 s. Samples were incubated at room temperature for 5 min and then centrifuged at 12,000g for 5 min. 750 µl of the supernatant were mixed with 750 µl ethanol and RNA isolated using the Direct-zol RNA Miniprep Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. This Kit includes a DNAse treatment step. The concentration and purity were measured using the NanoDrop ONE (Thermo Scientific).

Synchronous populations of wildtype or rsp-2(ok639) L1 animals were generated for RNA extraction by hypochlorite treatment. Approximately 60,000 animals were harvested from 6 independent populations per strain. After washing in S-basal, populations were subjected to freeze-thaw prior to sonication (10 s max, 3 times) using a 130 Watt 20 kHz Ultrasonic Processor and 2 mm stepped microtip. Samples were retained on ice for a maximum of 30 minutes. Total RNA was extracted using RNAzol RT (Sigma-Aldrich) following manufacturers guidelines. Total RNA pellets were resuspended in water and reprecipitated with lithium chloride (Invitrogen) to remove contaminants and residual DNA. Samples were resuspended in 10 mM Tris, and concentration was measured using a Nanodrop spectrophotometer. 1 ug of RNA was converted to cDNA using the Maxima H-minus First Strand cDNA Synthesis kit (ThermoFisher Scientific) which includes a dsDNAase degradation step. Samples were diluted in 10 mM Tris to 10 ng/uL.

Total RNA from bacterial strains was extracted using the hot phenol procedure (Aiba et al., 1981 (link)) or the RiboPure™ Bacteria Kit (Invitrogen, ON, Canada, Cat. No. AM 1925) following the manufacturer’s recommendations. OMV-derived RNAs were isolated with RiboPure™ Bacteria Kit or RNAzol RT (Sigma, MO, United States, Cat. No. R4533) reagents following the manufacturer’s recommendations. RNAzol RT kit allowed selective isolation (enrichment) of small RNAs (<200 nt). All RNA samples were subjected to treatment with DNase-I when applicable, quantified with the NanoDrop™ 2000 Spectrophotometer (Thermo Scientific™, Cat. No. ND-2000) and saved at −80°C before downstream applications.

Total RNA was extracted from the cells using RNAzol RT according to manufacturer’s instructions (Sigma Aldrich, St. Louis, MO, USA) and quantified using a Nanodrop Lite Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). 100 ng of RNA were reverse transcribed to cDNA using a High Capacity RNA-to-cDNA kit (Applied Biosystems) for further use in quantitative RT-PCR (qRT-PCR). Transcripts encoding for EGR1 and GAPDH were measured by TaqMan qRT-PCR with PerfeCTa FastMix II Low ROX (QuantaBio, Beverly, MA, USA). TaqMan gene expression assays for each transcript (Hs00152928_m1 (EGR1) and Hs99999905_m1 (GAPDH)) were analyzed in a 7500 Fast Real-Time PCR System (Applied Biosystems). The level of target mRNA was estimated by relative quantification using the 2^−ΔΔCt method and GAPDH as housekeeping gene. Quantification of each cDNA sample was tested in triplicate. A corresponding non-reverse transcriptase reaction was included as a control for DNA contamination.

The expression of genes was assessed in roots using quantitative real-time polymerase chain reaction (qRT-PCR). Total RNA was extracted and purified using an RNAzol RT (Sigma-Aldrich) according to manufacturer’s instructions. RNA samples were DNase treated with DNase I (Thermo Scientific^™). Total RNA (200 ng) was used to generate first-strand complementary DNA (cDNA) by RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific^™) with oligo(dT)18 Primer in a total volume of 35 μl, as is described in the manufacturer’s guideline. qRT-PCR was performed in a CFX Connect^™ Real-Time PCR System. iTaq^™ Universal SYBR^® Green Supermix (Bio-Rad) was used as the basis for the reaction in a total volume of 12 μl (6-μl PCR Supermix, 1-μl primer, 4-μl H₂O, and 1-μl cDNA).
Table S1 shows the primer pairs used to amplify the genes.
For the normalization of the expression levels, housekeeping genes EF1α and PP2Acs were used as a reference genes; cDNA from untreated material was used as a reference sample.