Acid phenol

HEK293 cells were plated at 10⁶ per 10 cm plate 24 h before transfection with 30 nM siRNA (control siRNA #3 from Dharmacon; specific siRNA from Ambion: CNOT1—ID no. S22844, TNRC6A—ID no. S26154, TNRC6B—ID no. S23060) and Dharmafect 1. Forty-eight hours after transfection, cells were harvested by scraping into ice cold PBS, spun down, and directly extracted in Trireagent (Invitrogen) followed by acid phenol (Ambion) extraction. Four micrograms of RNA was subjected to library preparation using the TruSeq Stranded mRNA library preparation kit followed by NextSeq500 High Output 75 cycle sequencing. The TNRC6A knockdowns were sequenced in duplicate, and CNOT1 knockdown sequencing performed in quadruplicate.

The FHV in vitro replication assay (IVRA) described here was adapted from Short et al. [36 (link)]. Briefly, 8 µL of CMP was added to a replication buffer (50 mM Tris, pH 8.0; 18 mM MgCl₂; 30 mM KCl; 16 mM NaCl; 250 mM sucrose; 10 µg/mL actinomycin D; 0.2 U/µL RiboLock; 1 mM ATP/GTP/UTP; 10 µM CTP; 5 µCi of CTP[α-32P] (specific activity, 3000 Ci/mmol, Perkin Elmer) to a final volume of 50 µL. The reaction was incubated for 90 min at 30 °C and the unincorporated nucleotides were removed using Micro Bio-Spin P-30 gel columns, Tris buffer, RNase-free (Bio-Rad, Berkeley, CA, USA). RNA isolation was performed as described by Scholte et al. [37 (link)]. In brief, acid phenol (Ambion) was used for the extraction following which RNA was precipitated with isopropanol, washed with 75% ethanol, and re-suspended in 20 µL of 1 mM sodium citrate (pH 6.4). A denaturing mixture (67% formamide, 23% formaldehyde, 6.7% glycerol, 13 mM MOPS (pH 7.2), 6.7 mM NaAc, 2.7 mM EDTA, 0.07% SDS, and 0.03% bromophenol blue) [37 (link)] was added to the RNA samples, incubated at 75 °C for 15 min, briefly vortexed and put on ice. RNAs were separated in a 1% denaturing formaldehyde agarose gel for 2 h at 100 V. The gel was dried and exposed to a phosphorimaging plate. Detection was performed using Typhoon Trio (GE Healthcare Life Sciences).

Stem-loop qRT-PCR was done to detect the levels of miR530-5p in SN and LGR seed developmental stages (S1–S5). Total RNA was isolated from S1–S5 stages and flag leaf of SN and LGR as mentioned above. The total RNA was purified using acid phenol (Ambion®; Naugatuck, CT, USA) as per manufacturer’s protocol. For stem-loop qRT-PCR, miR530-5p specific cDNA was synthesized using its specific stem-loop reverse transcription primer (miR530-5p SL primer) (Additional file 2: table 17). This cDNA was synthesized from 200 ng of total RNA by using Superscript™ III first-strand cDNA synthesis kit (Invitrogen™; USA) according to the manufacturer’s protocol. This cDNA was used for qRT-PCR assay using miR530-5p forward primer and a universal reverse primer. The conditions for qRT-PCR were maintained same as mentioned previously. snRNA U6 was used as an internal control. Three biological replicates were used for the assay and significance was calculated using Student’s t test with p value˂0.001 denoted as double asterisk (**).

To isolate miRNA-containing RNAs, cells were lysed and homogenized with Acid Phenol (Ambion®, Life Technologies, Darmstadt, Germany) and RNA was isolated using the miRVana RNA isolation kit (Ambion®, Life Technologies, Darmstadt, Germany) according to the instructions provided by the manufacturer. RNA concentration was measured using the Nanodrop 1000 (Thermo Scientific, Waltham, USA) and RNA integrity was assessed using the Bioanalyzer (Agilent, Santa Clara, USA).
cDNA synthesis and primer design (Tab. 2) for the analysis of miRNA expression, profiling by miQPCR was carried out as previously described31 (link). In brief, 10 ng of total RNA was used for cDNA synthesis and a fraction of the synthesized cDNA (equivalent to 1% of final cDNA volume) was used for individual qPCR assays. Primer design was performed as described in31 (link), miRNA specific primers used in this study are listed in Table 2. qPCR assays were performed in a 96 well format and run on a ViiA7 thermocycler (Applied Biosystems®, Life Technologies, Darmstadt, Germany) using Bryt^® Green (Promega, Mannheim, Germany) miRNA amplification parameters were as follows: 95 °C for 10 minutes (1 cycle), 95 °C for 15 seconds and 60 °C for 35 seconds (50 cycles), followed by melting curve analysis. qPCR data were analyzed by qBase software v.1.3.546 (link).

Sulfolobus solfataricus exosome was purified as described previously (Witharana et al. 2012 (link)). 200 nM RNA labelled with a 5′-fluorescein (RNA-FAM) was incubated with wild-type SSB (concentrations from 0 to 480 µM) for 5 min at room temperature in 20 mM HEPES (pH 7.9), 0.1 mM EDTA, 60 mM KCl, 8 mM MgCl₂, 2 mM DTT, and 10 mM K₂HPO₄. To each aliquot, 0.5 µl S. solfataricus Rrp41-Rrp42 hexameric ring and 0.4 µl Rrp4 protein were added. The total volume of each aliquot was 10 µl. The reaction was left to incubate at 60 °C for 1 h. 10 µl of each sample was added to acid phenol (Ambion) and mixed thoroughly, then spun at 13,000 rpm for 1 min. 5 µl from the resulting supernatant was added to 5 µl formamide (Promega) and loaded on to a denaturing gel (25% polyacrylamide, 7 M urea, 300 µl of ammonium persulfate (APS), and 30 µl of TEMED, 5 ml TBE, 5 ml water, total volume 50 ml) run at 85 W with a temperature threshold of 50 °C for 2.5 h. The gel was scanned using Fuji FLA5000 phosphorimager and analysed using the ImageJ software.