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9 protocols using dsrna ladder

1

Ubiquitination of TRIM25 by E2 enzymes

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TRIM25 constructs were incubated at 37 °C with E1 (100 nM), E2 (1 μM Ube2D3 or 0.28 μM Ube2N/Ube2V2), and ubiquitin (40 μM) in reaction buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5 mM TCEP, 5 mM ATP, 10 mM MgCl2). Reactions were stopped by addition of SDS-PAGE sample buffer and boiling for 10 min. Immunoblots were performed with anti-Ub (1:2,000; P4D1, Santa Cruz Biotechnology) and anti-FLAG M2 (1:5,000; Sigma). Experiments in Fig. 2 used RNase A (Qiagen), DNase I (Sigma), dsRNA ladder (NEB), and 100-bp dsDNA ladder (NEB).
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2

RNA Extraction and Purification Protocol

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Tris base, ethylenediaminetetraacetic acid (EDTA), potassium acetate (KoAc), sodium acetate (NaOAc) and lysozyme were purchased from Sigma-Aldrich. TAE running buffer (50×) was purchased from Omega Bio-Tek. Sodium hydroxide (NaOH) was obtained from EM Science. Boric acid was purchased from JT Baker. Sodium dodecyl sulfate (SDS) was purchased from Mallinckrodt Baker. RNase A/T1 Cocktail Mix and RNase V1 were purchased from Invitrogen/Life Technologies. RNase If, 2-log DNA ladder, and dsRNA ladder were purchased from New England Biolabs. RNase A was obtained from Thermo Scientific.
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3

Nucleic Acid Protection Assays with NS4

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Nucleic acid protection assays were based on the method described by Belhouchet et al. [32 (link)]. For the DNA protection assays, 1 μg of dsDNA (BenchTop pGEM DNA Marker, Promega) in DNaseI buffer was mixed with 500 ng of purified recombinant NS4 in a volume of 9 μL and incubated at room temperature for 20 minutes. Thereafter, 2 units of DNase I (New England Biolabs) were added to the reaction, which was incubated at 37°C for 30 minutes. The enzyme was inactivated by heating the reaction to 99°C for one minute. The reactions were analysed with agarose gel electrophoresis using agarose gels containing 2% (w/v) agarose and 0.5 μg/mL ethidium bromide. RNA protection assays were performed in the same manner with the same quantities of dsRNA (dsRNA Ladder, New England Biolabs), NS4 protein and RNase III (ShortCut RNase III, New England Biolabs), but with the addition of 20 mM MnCl2 to the reaction. After incubation, the enzyme was inactivated by the addition of 50 mM EDTA and the reactions were analysed with 3% agarose gel electrophoresis.
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4

Monitoring tRNA Cleavage in Fibroblasts

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Normal human fibroblasts were seeded at a density of 900,000 cells per 10 cm dish, grown for 24 h, and treated with 50 μM NaAsO2 for indicated times. Total RNA was extracted using RNAzol RT (Sigma, R4533) following the manufacturer’s protocol. A total of 1 μg RNA was then separated using precast 15% Novex TBE-Urea gel (ThermoFisher, EC6885BOX), and the gels were stained with SYBR Gold (ThermoFisher, S33102) diluted 1:10,000 in 1× TBE for 30 min prior to imaging (BioRad). dsRNA ladder (NEB, Whitby, ON, Canada, NO363S) was used as a size marker. To assess tRNA cleavage following either TRNT1 or angiogenin knock-down, cells were reverse-transfected with appropriate siRNA (see above) for 48 h prior to NaAsO2 treatment.
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5

Reagents for RNA-related Experiments

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The following reagents were purchased: mouse mAb J2 (Scicons); Alexa Fluor 488-goat anti-mouse IgG secondary antibody (Molecular Probes, Life Technologies); Phi6 dsRNA (Finnzymes, Thermo Fisher Scientific); dsRNA ladder (New England Biolabs); High MW poly (A:U) and poly (I:C) (InvivoGen); GeneRuler 1 kb DNA Ladder (Thermo Fisher Scientific); Silencer negative control 1 siRNA (Ambion, Life Technologies); Strep-Tactin conjugated to horseradish peroxidase (IBA Lifesciences); pNPP (Sigma-Aldrich), mMESSAGE mMACHINE T7 transcription kit (Ambion, Life Technologies), Lumi-Light western blotting substrate (Roche Diagnostics).
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6

Detecting dsRNA by Dot Blot Assay

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Crude IVT reactions or purified IVT RNA samples were spotted onto positively charged nylon membranes (Nytran SC, Sigma-Aldrich). The membranes were blocked in 5% (w/v) nonfat dried milk in TBS-T buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.1% [v/v] Tween-20). For the detection of dsRNA, the membranes were incubated with J2 anti-dsRNA antibody (1:5000; Scicons) at 4°C overnight. The blots were probed with IR Dye -680 or -800 conjugated secondary antibodies (Cell Signaling Technologies). dsRNA ladder (New England Biolabs) and poly(I:C) (Invivogen) were used as positive controls.
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7

Molecular Biology Reagents and Standards

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Tris base was purchased from J. T. Baker and glacial acetic acid was from Mallinckrodt Chemicals. Ethylenediaminetetraacetic acid (EDTA)-free acid, EDTA-disodium salt and Omnipur agarose were obtained from EMD Chemicals, Inc. Boric acid was purchased from Sigma-Aldrich and ethidium bromide (EtBr) was from Shelton Scientific, Inc. The 2-Log DNA ladder, 1 Kb DNA ladder, dsRNA ladder and siRNA ladder standards were purchased from New England Biolabs.
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8

Nucleic Acid-Lipid Complex Assessment

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To assess the extent of complex formation between nucleic acids and lipids, 1.5–2% agarose gels were prepared using 1 µL of GelRed in 1× Tris-Acetate-EDTA (TAE) buffer. All nanoparticle samples (PEG-LNP, HiPerFect) and the controls, including dsRNA Ladder (New England Biolabs, N0363S), as well as pure nucleic acids, were applied in comparable concentrations to the gel. Finally, the samples were treated with 1% Triton X-100 to release the miRNA. In some cases, endonuclease was added to induce miRNA digestion.
The gel was placed into an electrophoresis tank (Embi Tec, RunOne Elecrophoresis cell; Thermo Fisher Scientific, Vienna, Austria) and covered with 1× TAE buffer. The power supply voltage was set to 75 V. After running for a duration of 35–40 min, the gel was transferred onto the UV-tray and scanned with BioRad GelDoc EZ Imager (BioRad Laboratories, Hercules, CA, USA). Finally, data processing and analysis were performed using the Image Lab Software (BioRad, version 6.1).
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9

Phage Genome Nucleic Acid Composition Analysis

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Phage genomes were isolated by the phenol/chloroform method described [20 (link)]. To determine the nucleic acid composition, 1 μg of each genome was incubated with DNase I or RNase A (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions, separated on a 1% agarose gel and imaged. To determine ds or ssRNA in CAP3, RNase If (New England BioLabs, Ipswich, MA, USA) was added per manufacturer’s instructions to the ssRNA ladder (New England BioLabs), dsRNA ladder (New England BioLabs) and CAP3 genomic RNA. The reaction was stopped with 0.1% sodium dodecyl sulfate (final volume) and separated on a 2% agarose gel and imaged.
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