Turbo dnase 1

Total RNA was purified from approximately 2×10⁸ yeast cells with the RiboPure-Yeast^TM Kit (AM1926, Invitrogen) according to the supplied manual. For treatment with Turbo DNase I (AM1917, Invitrogen), 20 µl of nucleic acid sample were incubated with 5 µl DNase I buffer and 2 µl of DNase I (8U) for 60 min (37 °C). After 30 min, 8 more units of DNase I were added. The reaction was stopped by adding 0.1 x DNase Inactivation Reagent and vortexing. Samples were incubated for 5 min at RT and centrifuged (3 min, 16 100 g). The supernatant was transferred to a fresh 1.5 ml tube, the concentration of nucleic acids was determined with a Nanodrop (NanoDrop One, Thermo Fisher) and 2 µg were used for reverse transcription with SuperScript II^TM (18064014, Invitrogen) according to the manufacturer’s instructions. RNaseOUT^TM (10777019, Invitrogen) was added to the reaction. DNA contamination was assessed via PCR with primers amplifying UBC6. qPCR was performed in a 20 µl reaction with KAPA SYBR® FAST qPCR Master Mix (KM41001, Merck) according to the manufacturer’s instructions in a Rotor-Gene Q (QIAGEN) PCR cycler. All primers for qRT-PCR are listed in Supplementary Table 8. Data are represented as fold changes to WT after COQ mRNA levels were calculated relative to UBC6 mRNA levels with the comparative CT method (∆∆CT)^{75 (link)}.

Total RNA was extracted from roots using the Plant/Fungi Total RNA Purification Kit (Norgen Biotek Corp., Thorold, ON, Canada), treated with TURBO DNase I (Invitrogen) and assessed for purity and quality. cDNA was prepared from 500 ng of RNA using the SuperScript IV VILO Master Mix (Invitrogen). qRT‐PCR was performed using three to five biological and three technical replicates, on a CFX384 Real‐Time PCR Detection System (Bio‐Rad, Mississauga, ON, Canada) using the SensiFAST SYBR No‐ROX kit (Bioline, Memphis, TN, USA) under the following conditions: 95°C for 3 min followed by 40 cycles of 95°C for 5 s, 60°C for 15 s and 72°C for 15s. Expression levels were normalized against three reference genes (UBQ, PP2A and ATP‐s) as previously described (Held et al., 2014). Primer sequences used for the qRT‐PCR expression analyses are listed in Table S1.

Tissue samples (10 mg) were homogenized in a Micro-Dismembrator U using steel beads (Sartorius, Göttingen, Germany). Total RNA isolation from PBMC, BDM and lung samples was performed by chloroform extraction and isopropanol precipitation. RNA was treated with TurboDNaseI (Invitrogen, Carlsbad, CA, USA) and purified by extraction with phenol acid, chloroform, and ethanol precipitation. Total RNA (1 µg) was retrotranscribed to cDNA with Transcriptor First Strand cDNA Synthesis (Roche, Basel, Switzerland) using oligo-dT primers.

RNA was isolated from S. coelicolor cultures cultivated in 5 ml YEME/TSB liquid medium for 24 h. Mycelia were collected by centrifugation, frozen and stored at -70°C for subsequent RNA isolation. RNA was isolated using the RNeasy Mini Kit (Qiagen) following the manufacturer's instructions, digested with TURBO DNase I (Invitrogen) and checked for chromosomal DNA contamination using PCR. A total of 500 ng of RNA was used for cDNA synthesis using the Maxima First Strand cDNA synthesis kit (Thermo Fisher Scientific) in a final volume of 20 μl. The obtained cDNA was diluted to 100 μl and directly used for quantitative PCRs performed with PowerUp SYBR Green Master Mix (Applied Biosystems). The relative level of hupA was quantified using the comparative ΔΔCt method, and the hrdB gene was used as the endogenous control (StepOne Plus real-time PCR system, Applied Biosystems).

A549 cells were infected with rRSVflag(2)L at an MOI = 3. At 20 hpi, cells were lysed in RIPA lysis buffer and genomic DNA was digested with TURBO DNaseI (Invitrogen, AM2238). 5% of the sample was run on a tris-glycine gel and was transferred to a nitrocellulose membrane using XCell II blot module (Invitrogen) for 2 hrs at 30V in a transfer buffer containing 10% methanol. Blot was analyzed with primary antibody ANTI-FLAG(R) M2 (Sigma-Aldrich, F3165) at a dilution of 1:500 and secondary antibody IRDye 800CW anti-Mouse IgG (Licor, 26–32212) at a dilution of 1:20,000.