The template DNAs for in vitro transcription of Int2-1, Int2-2, Int2-3, Int2-1-2-1, Int2-1-2-2, Int2-1-2-2a, Int2-1-2-2b and Int2-1-2-2pSL RNA were amplified with KOD-Plus-Neo DNA polymerase using the Ex1-(Int2)-Ex4-GFP minigene as a template for Int2-1, Int2-2, Int2-3, Int2-1-2-1, Int2-1-2-2, Int2-1-2-2a and Int2-1-2-2b and Int2pSL for Int2-1-2-2pSL, respectively. Supplementary Table S1 lists the primer sets. Amplified DNA was separated by 1% agarose gel electrophoresis and purified from gel pieces using the QIAquick Gel Extraction kit. In vitro transcription was performed with 0.2 pmol of the template DNA using CUGA® 7 in vitro transcription kit (NIPPON GENE). Transcripts were collected with the RNeasy Mini kit (Qiagen) (Int2-1, Int2-2, Int2-3) or isopropanol precipitation (Int2-1-2-1, Int2-1-2-2, In2-1-2-2a, Int2-1-2-2b, Int2-1-2-2pSL) and purified using reverse-phase liquid chromatography. Biotin (BT) was added to the 5′ end of RNA using the 5′ EndTag Nucleic Acid End Labeling System and Biotin (Long Arm) Maleimide (Vector Labs). BT-RNA was collected by ethanol precipitation.
Cuga 7 in vitro transcription kit
Manufactured by Nippon Gene
Sourced in Japan
The CUGA 7 in vitro Transcription Kit is a lab equipment product developed by Nippon Gene. The kit facilitates the in vitro transcription of RNA from DNA templates.
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Lab products found in correlation
Rneasy mini kit, by Qiagen (3 mentions)
Pgem 3z plasmid vector, by Promega (2 mentions)
Rnase free dnase set, by Qiagen (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Biotin long arm maleimide, by Vector Laboratories (1 mentions)
Biotin 16 utp, by Merck Group (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
T7 rna polymerase, by Takara Bio (1 mentions)
Imagequant, by Cytiva (1 mentions)
Amersham typhoon rgb, by Cytiva (1 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (1 mentions)
Rneasy minelute cleanup kit, by Qiagen (1 mentions)
Amersham typhoon scanner, by Cytiva (1 mentions)
Multi gauge software, by Fujifilm (1 mentions)
L glutamine, by Nacalai Tesque (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Nacalai Tesque (1 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
15 protocols using cuga 7 in vitro transcription kit
1
In Vitro Transcription of Intron Variants
2
Synthesis and Characterization of Biotin-Labeled Lionheart RNA
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To obtain the biotin-labeled full-length or truncated Lionheart, we used CUGA® 7 in vitro Transcription kit (NIPPON GENE). When the biotin-labeled RNAs were synthesized, biotin-16-UTP (Sigma-Aldrich) was added, as the ratio of biotin-16-UTP to usual UTP was 3:2. The PCR product containing T7 promoter was employed as the template. The quantity and the quality of the synthesized RNAs were confirmed by NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific) and Agilent 2100 bioanalyzer (Agilent Technologies). The RNAs were saved at −80 °C until use. To identify the Lionheart-specific-binding proteins, we used the biotin-labeled Lionheart antisense (AS) as the control.
3
Partitivirus Coat Protein Expression
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The CP gene from each partitivirus was cloned into the CMV-H1 vector57 (link), respectively, to be expressed as RNA4A fragments derived from cucumber mosaic virus (CMV). The DNA fragment for RNA4A carrying the partitivirus CP sequence was amplified by PCR using primer pair CM95-4A-5-T7/SSV-12-3, then used as template for in vitro transcription by T7 RNA polymerase (Takara Bio, Shiga, Japan). For inducing RNA silencing against GFP, GFP dsRNA was synthesized as follows: DNA fragments containing partial GFP sequences were PCR-amplified using primer pair EGFP-5-T7-330/EGFP-3-T7-330 with the pMF280-EGFP (GFP-expressing plasmid for fungi) as a template, and the obtained PCR products were transcribed in vitro in both directions using the CUGA 7 in vitro transcription kit (NIPPON GENE CO., LTD, Tokyo). pMF280-EGFP was kindly provided by Dr. Teruo Sone (Hokkaido University, Sapporo, Japan). The plasmid constructs for the RSS activity assay using Rhizoctonia protoplasts was shown in Supplementary Fig. S11 .
4
In Vitro Protein Synthesis Assay
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RNA templates produced by CUGA®7 in vitro transcription kit (Nippon Gene). The PURE system (PUREfrex, Gene-Frontier) reaction was carried out at 37 °C for 2 h in the presence or absence of 1 µM IbpA, which included Cy5 labeled tRNAfMet. After protein synthesis, SDS-sample buffer (0.125 M Tris-HCl (pH 6.8), 10% (v/v) 2-mercaptoethanol, 4% (w/v) SDS, 10% (w/v) sucrose, 0.01% (w/v) bromophenol blue) was added and incubated at 95 °C for 5 min. The samples were then separated by SDS-PAGE and detected using a fluorescence imager (Amersham Typhoon RGB, Cytiva) at the 633-nm wavelength. The band intensity was quantified with image analysis software (Image quant, Cytiva).
5
RNA-Protein Interaction Profiling
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The RNA pull-down assay was performed using RiboTrap Kit (MBL), according to the manufacturer’s instructions. Briefly, the cDNA fragments corresponding to the coding region or 3′-UTR of mouse Inka2 cDNA were subcloned into a pGEM-3Z plasmid vector (Promega) for in vitro transcription. The BrU–labeled RNA was prepared using a CUGA 7 in vitro Transcription Kit (Nippon Gene, Japan). Purified BrU-labeled RNA (50 pmol each) was bound on the Protein G Plus-Agarose beads conjugated to an anti-BrdU monoclonal antibody. Cytoplasmic extract prepared from N2a cells (approximately 1 × 108 cells) according to the manufacturer’s instruction (MBL) was precleared by the treatment with Protein G beads and then incubated with the BrU-labeled RNAs on antibody conjugated beads for 2 h at 4°C. After washing the sample beads four times with the low-ionic strength wash buffer I (+) supplemented with 1.5 mM DTT, BrU-RNA/protein complexes were eluted with 50 μL PBS containing BrdU by immunoblotting.
6
SARS-CoV-2 RNA Fragment Synthesis
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The SARS-CoV-2 sequence used herein was obtained from NCBI (NCBI Reference Sequence: NC_045512.2) and the GISAID database (www.gisaid.org). Five DNA fragments (wild type, L452R, T478K, E484K, and E484Q mutants; 600-1000 bp in length) with a 5′ T7 upstream promoter sequence were obtained from Eurofins Genomics KK (Tokyo, Japan).
RNA fragments were synthesized by in vitro T7 transcription (CUGA 7 In Vitro Transcription Kit; Nippon Gene Co. Ltd., Tokyo, Japan), in accordance with the manufacturer's instructions. RNA fragments were purified on spin columns (RNeasy Mini Kit; Qiagen GmbH, Hilden, Germany). Each eluent was treated with DNase I (RNase-Free DNase Set; Qiagen) and repurified on a new spin column. Synthesized single-stranded RNA fragments were used as RT-PCR amplification templates.
RNA fragments were synthesized by in vitro T7 transcription (CUGA 7 In Vitro Transcription Kit; Nippon Gene Co. Ltd., Tokyo, Japan), in accordance with the manufacturer's instructions. RNA fragments were purified on spin columns (RNeasy Mini Kit; Qiagen GmbH, Hilden, Germany). Each eluent was treated with DNase I (RNase-Free DNase Set; Qiagen) and repurified on a new spin column. Synthesized single-stranded RNA fragments were used as RT-PCR amplification templates.
7
In Vitro Transfection of siRNA
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Set siRNA and negative control (Scramble) RNA were produced using the CUGA7 in vitro transcription kit (Nippon Gene, Tokyo, Japan). The siRNA sequences are as follows: Set siRNA 5′-GGATGAAGGTGAAGAAGAT-3′ and Scramble 5′-CAGTCGCGTTTGCGACTGG-3′. siRNA transfection was performed using Lipofectamine RNAiMAX transfection reagent (Invitrogen).
8
Generating Recombinant SAFV-3 Viruses
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pSAF/UnaG and pSAF/GFP were linearized with NotI, and infectious RNA transcripts were synthesized using a CUGA 7 in vitro Transcription Kit (NIPPON GENE) to avoid termination of RNA transcription at the human preproparathyroid hormone (PTH) signal in the genome of SAFV-3 (JPN08-404), as described previously [8 ]. To generate recombinant viruses, BHK-21 cells were seeded at a density of 3.0 × 105 cells per 35-mm dish. On the following day, the transcripts (10 µg) were transfected into the cells using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions. At 20 h post-transfection, cells and supernatants were collected, and viruses were prepared by three freezing and thawing cycles to release virions, followed by centrifugation to remove cell debris. These clarified supernatant stocks from BHK-21 cells were designated as passage 0 (P0). Thereafter, virus was propagated in HeLa cells, and the lysate was prepared by three freezing and thawing cycles. Viral titers were determined by a standard plaque assay on HeLa cells.
9
Preparation of mRNA and DNA Templates
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For use as a template in the PURE system for the FCS measurements in Figure 1 and Supplementary Figure S1 and TIRFM observations, the gatY gene was inserted into the pET21 vector with the external sequence including the HA-tag (MSYPYDVPDYAH) at the N-terminus. The genes for the time-course observation in Figure 2 and Supplementary Figure S2 (gatY1-284, araA1-500, nuoC1-600, uxaC1-470, xylA1-440, yfbQ1-405, dadA1-432, dapA1-292, fadA1-387, and pmbA1-450) were amplified by PCR from the plasmid used in the previous study (Fujiwara et al., 2010 (link)), harboring each gene located downstream of the tac promoter. Consequently, the T7 promoter sequence was attached at the 5′ UTR region by the primer. Truncated DNA fragments were prepared by PCR amplification with appropriate primers, as listed in Supplementary Table S1 . The DNA fragment was then transcribed with a CUGA7 in vitro transcription kit (Nippon Gene, Japan). After the transcription, the mRNA was purified with an RNeasy MinElute Cleanup Kit (Qiagen, Germany). The purified mRNA was quantified by the absorbance at 260 nm. When DNA was used as a template instead of mRNA, the DNA fragment used for the transcription was employed.
10
Synthetic SARS-CoV-2 RNA Fragment Synthesis
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Because each synthetic DNA fragment possesses a 5′ T7 upstream promoter sequence, the SARS-CoV-2 RNA fragments were synthesized by in vitro T7 transcription (CUGA 7 in vitro Transcription Kit; Nippon Gene Co. Ltd.) according to the manufacturer's instructions. Synthetic RNA fragments were purified on spin columns (RNeasy Mini Kit, Qiagen). After purification, each eluent was treated with DNase I (RNase-Free DNase Set; Qiagen) and repurified on a new spin column. Synthetic single-stranded RNA fragments (250 ng/lane) were separated by non-denaturing agarose gel electrophoresis using RNA loading buffer and RNA ladder (DynaMarker RNA High for Electrophoresis; BioDynamics Laboratory Inc., Tokyo, Japan) according to the manufacturer's instructions. Images were captured by FAS-IV.
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