Nucleospin rna clean up

The samples were removed from RNAlater and cut into smaller pieces. One ml of Trizol (Invitrogen, Carlsbad, USA) was added to each sample and the tissue was homogenized in a glass tissue grinder. After addition of 0.2 ml of chloroform and centrifugation of the sample, 100 µl of the upper aqueous phase was mixed with 350 µl lysis buffer from the NucleoSpin® RNA Plus Kit (Macherey Nagel, Düren, Germany). RNA was then isolated according to the manufacturer’s instructions as described in the user manual of the kit. rDNase treatment and subsequent clean-up (NucleoSpin RNA Clean-up, Macherey Nagel) was performed according to the manufacturer’s protocol to ensure RNA purity.
Sample preparation and sequencing were performed at SciLifeLab Uppsala, SNP&SEQ Technology Platform. RNA concentration and integrity were checked by Fragment Analyzer (Agilent, Santa Clara, USA) before sequencing libraries were prepared from 500 ng total RNA using the TruSeq Stranded mRNA Library Preparation Kit including polyA selection (Illumina Inc., San Diego, USA) resulting in insert sizes of approximately 140 bp. Three biological replicates per condition were sequenced using Illumina NovaSeq S1 flow cells and 100 bp paired end v1 sequencing chemistry, resulting in 36 transcriptomes.

Total RNA was extracted from healthy wild type (controls), BRB and ARB transgenic, and TMVi wild type tobacco plants by using TRIsure reagent (Bio line, UK) according to manufacturer's instructions. The extracted total RNA was purified with the RNA purification kit (Nucleospin RNA clean-up, Macherey-Nagel) and then subjected to DNaseI treatment (Promega RQ1 RNase free-DNaseI) according to the manufacturer's recommendations. Subsequently, the total RNA was concentrated with Amicon Ultra-0.5 centrifugal filter devices. The cDNA labeling, and Agilent 4×44k microarray hybridization were done according to the manufactures instructions (Center for Biotechnology, Turku, Finland) and the raw numerical data handling and its statistical analysis were done by using Chipster (CSC, Finland) program [81] (link) as previously described [41] (link).

For production of double-stranded RNA (dsRNA), oligos with tails containing T3 and T7 promoters were used to amplify regions from N2 genomic DNA or cDNA. Primers used for dsRNA production are listed in S3 Table. PCR reactions were cleaned (NucleoSpin Clean-up, Macherey-Nagel) and used as templates for T3 and T7 transcription reactions (MEGAscript, Invitrogen). Transcription reactions were cleaned (NucleoSpin RNA Clean-up, Macherey-Nagel) and complementary single-stranded RNAs were annealed in soaking buffer (3x soaking buffer is 32.7 mM Na₂HPO₄, 16.5 mM KH₂PO₄, 6.3 mM NaCl, 14.1 mM NH₄Cl). dsRNAs were delivered by injecting L4 hermaphrodites, and animals were processed for live imaging after incubation at 20°C for 24 h or 48 h for partial and penetrant depletions, respectively.

Pooled samples of three to five leaves randomly selected from the canopy were used for RNA extractions from the symptomatic or from the asymptomatic control seedlings. Total RNAs were isolated using the InviTrap^® Spin Plant RNA Mini Kit (STRATEC Molecular, Germany), followed by removal of remaining DNA with rDNase according to the supplier protocol (Macherey-Nagel, Germany) and RNA purification using NucleoSpin^® RNA Clean-up (Macherey-Nagel, Germany). Ribosomal RNA depletion was performed using the RiboMinus Plant Kit for RNA-Seq (Invitrogen). One to two micrograms of RiboMinus RNA of each sample were used for cDNA synthesis with the Maxima H Minus double-stranded cDNA synthesis Kit (Thermo Scientific) primed with random hexamers. One to two micrograms purified double-stranded cDNA from each sample were sent to BaseClear (Netherlands) for RNA-Seq analysis with the Illumina HiSeq2500 system and 100 bp-long paired-end sequence reads corresponding to 50–100 Mb data/sample were generated.

Total RNA was extracted from a 50 mg specimen using 750 µl Trizol LS reagent (Invitrogen, Cat. N°. 10296-010), treated with DNase I (QIAGEN, Cat. N°. 79254) and then purified using NucleoSpin RNA Clean-up (Macherey-Nagel, Cat. N°. 740948.50). Total RNA was eluted with 50 µl DNase I-free water. Approximately 20 to 30 µg were recovered per biopsy.
RiboMinus RNA (ribosomal RNA-depleted RNA) was purified from 5 µg total RNA using the RiboMinus Eukaryote Kit for RNA-Seq (Invitrogen, Cat. N°. A10837-08).
RNA Integrity was assessed using an Agilent Bioanalyzer.

For production of double-stranded RNA (dsRNA), oligonucleotides with tails containing T3 and T7 promoters were used to amplify regions from genomic N2 DNA (gDNA) or cDNA (Table S2). PCR products were purified (NucleoSpin Gel and PCR Clean-up, Macherey-Nagel) and used as templates for T3 and T7 transcription reactions (MEGAscript, Invitrogen). Transcription reactions were purified (NucleoSpin RNA Clean-up, Macherey-Nagel) and annealed in soaking buffer (3× soaking buffer is 32.7 mM Na₂HPO₄, 16.5 mM KH₂PO₄, 6.3 mM NaCl, 14.1 mM NH₄Cl). dsRNAs were delivered by injecting L4 hermaphrodites. After injection, animals were incubated as follows before embryos were isolated for live-imaging: penetrant depletions, 48 h at 20°C; partial LIS-1 depletion (Fig. 1I,J), 24 h at 16°C; partial ROD-1 depletion (Fig. 3F,G), 24 h at 20°C; partial depletion of SPDL-1 and NDC-80 (Fig. 2D,E), 24 h at 16°C.