All RNAs other than HEV, FCV and radiolabeled transcripts (see RdRp assay protocol) were generated as follows. Five micrograms of linearized plasmid template were pretreated with RNAsecure (Thermo Fisher) and used in a 50 µL transcription reaction containing 5× transcription buffer (Thermo Fisher), 8 mM rNTPs, 1.25 units RiboLock RNase Inhibitor (Thermo Fisher), and 60 units of either T7 or SP6 RNA Polymerase (Thermo Fisher). After an overnight incubation at 30°C, 2 units RNA-free DNase (NEB) was added, and reactions left at 37°C for 30 min. RNA transcripts were precipitated by addition of LiCl and the resultant pellet washed in 70% ethanol before resuspension in RNase-free water. Capping of YFV and CHIKV replicon RNA transcripts was performed using the Vaccinia Capping System (NEB) according to manufacturer's recommendations. HEV and FCV transcripts were generated using the HiScribe T7 ARCA mRNA kit (NEB) using 1 µg of linearized plasmid template following manufacturer's instructions. The integrity of all RNAs was verified by running RNAs on a MOPS-formaldehyde agarose gel and visualizing product by SYBR Gold Staining (Thermo Fisher). When required, relative quantification of RNA species on the gels was achieved using a ChemiDoc XRS+ Imager (Bio-Rad).
Sp6 rna polymerase
Manufactured by Thermo Fisher Scientific
Sourced in United States, Lithuania
The SP6 RNA polymerase is an enzyme used in the in vitro synthesis of RNA. It recognizes and binds to the SP6 promoter sequence, initiating the transcription of RNA molecules from DNA templates.
Automatically generated - may contain errors
Lab products found in correlation
G 5 ppp 5 g rna cap structure analog, by New England Biolabs (3 mentions)
T7 rna polymerase, by Thermo Fisher Scientific (3 mentions)
Pcs2 ncas9n, by Addgene (2 mentions)
Pgem t easy vector, by Promega (2 mentions)
Rnaseout ribonuclease inhibitor, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Psfv1, by Thermo Fisher Scientific (2 mentions)
γ 32p atp, by PerkinElmer (2 mentions)
Dna purification kit, by Zymo Research (2 mentions)
14c leucine, by PerkinElmer (2 mentions)
3h leucine, by PerkinElmer (2 mentions)
Chemidoc xrs imager, by Bio-Rad (1 mentions)
Hiscribe t7 arca mrna kit, by New England Biolabs (1 mentions)
Rnasecure, by Thermo Fisher Scientific (1 mentions)
Sybr gold staining, by Thermo Fisher Scientific (1 mentions)
Vaccinia capping system, by New England Biolabs (1 mentions)
Transcription buffer, by Thermo Fisher Scientific (1 mentions)
Ribolock rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Pgem t, by Tiangen Biotech (1 mentions)
Ribolock, by Thermo Fisher Scientific (1 mentions)
37 protocols using sp6 rna polymerase
1
In Vitro RNA Transcription Protocol
2
Tol2 Transposase mRNA Synthesis
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The Tol2 transposase mRNA was transcribed using the SP6 RNA polymerase (Thermo Scientific, Vilnius, Lithuania) and the NotI-digested pCS2FA-transposase vector (Tol2kit v1.2 #396), and including G(5’)ppp(5’)G RNA Cap Structure Analog (New England Biolabs, Ipswich, MA, USA), as described [47 (link)]. The transcribed RNAs were purified as described above.
3
Tol2-Mediated Zebrafish Transgenesis
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All zebrafish lines were generated through Tol2-mediated transgenesis [54 (link)]. Tol2 cDNA was transcribed by Sp6 RNA polymerase (#EP0131, ThermoFisher Scientific) after Tol2-pCS2FA vector linearization with NotI restriction enzyme (#IVGN0016, Anza, Invitrogen, ThermoFisher Scientific). All constructs were microinjected into the yolk of > 200 wild-type zebrafish embryos at the single-cell stage using the Tol2 transposase system for germline integration of the transgene according to Bessa et al. [55 (link)] with minor modifications. As a readout, GFP fluorescence was observed and its localization was annotated at 1, 2, and 3 days post fertilization (dpf) to evaluate enhancer activity, using GFP expression in the midbrain as transgenesis control.
As GFP reporter expression becomes masked by the pigmentation of the eye as the RPE develops, embryos were also treated with PTU to decrease eye pigmentation [56 (link)].
As GFP reporter expression becomes masked by the pigmentation of the eye as the RPE develops, embryos were also treated with PTU to decrease eye pigmentation [56 (link)].
4
Synthesis and Radiolabeling of Target RNAs
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NbAGO1-1L mRNA and CIRV p19 mRNA were synthesized in the presence of monomethylated cap analogue m7GP3G (Jena Biosciences, Germany) from SwaI-linearized plasmid constructs described by Gursinsky et al. (49 (link)) using SP6 RNA polymerase (Thermo). Transcripts encoding the firefly luciferase mRNA were generated by SP6 RNA polymerase from the XhoI-linearized plasmid pSP-luc(+) (Promega). Transcription reactions and subsequent treatment of the transcripts were performed by using standard procedures. To generate target RNAs for miRNAs 162, 168, and 403, cDNA fragments of the A. thaliana DCL1 ORF, the N. benthamiana AGO1-1L ORF, and the N. benthamiana AGO2 3′ untranslated region (UTR) containing the respective miRNA target site were amplified by PCR with the T7 promoter sequence included in the forward primer. Transcription of the target RNAs was performed from the PCR products by T7 RNA polymerase in the presence of 0.5 µCi/µl [α-32P]CTP (3,000 Ci/mmol) under standard conditions.
5
Whole-Mount In Situ Hybridization of elavl1a in Zebrafish Development
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The fertilized eggs, 2-cell embryos, high blastulae and gastrulae, 10-somite larvae, and 1-, 2-, and 3-day-old larvae were collected. A fragment of elavl1a was PCR-amplified using the primer pair P7 and P8 (Supplementary Data 1 ) and subcloned into vector pGEM-T (Tiangen Biotech, Beijing, China). Digoxigenin (DIG)-labeled elavl1a-specific antisense riboprobes were synthesized with linearized vectors (digested by Nco I enzyme) and Sp6 RNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA) through in vitro transcription. Whole-mount in situ hybridization was performed by the method of Thisse and Thisse33 (link). After staining, the embryos/larvae were observed and photographed under Nikon SMZ1000 stereomicroscope.
6
Synthesis of Capped RNA via In Vitro Transcription
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Plasmids were digested with MluI. Reaction mixtures for in vitro transcription consisted of 12.5 μl of water, 10 μl of 5X buffer for SP6 RNA polymerase, 5 μl 10 mM DTT, 2.5 μl of 10 mM synthetic cap analogue (m7G(5')ppp(5')G), 10 μl of rNTP solution (2.5 mM each rNTP), 10 μl (2 μg) of the linearized plasmid. To this mixture 1 μl of RNase inhibitor (RiboLock, ThermoFisher Scientific) and 2.5 μl of SP6 RNA polymerase (ThermoFisher Scientific) were added. The mixture was incubated at 41°C for 1 hour. Quality of RNA was examined by a gel electrophoresis in 1% agarose.
7
CRISPR-Cas9 sgRNA Generation for foxm1 Knockdown
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Nine sgRNA spacer sequences targeting foxm1 coding sequence were selected on Benchling (2018) [43 ] based on metrics from Doench and colleagues [44 (link)] and Hsu and colleagues [45 (link)]. Pairs of oligonucleotides where ordered (Sigma-Aldrich, Darmstadt, Germany) and annealed, followed by cloning in the BsaI-digested vector pDR274 (Addgene #42250), as described [46 (link)]. sgRNAs were transcribed using the T7 RNA polymerase (Thermo Scientific, Vilnius, Lithuania) and the HindIII-digested sgRNA expression vector as template. The Cas9 mRNA was transcribed using the SP6 RNA polymerase (Thermo Scientific, Vilnius, Lithuania) and the NotI-digested pCS2-nCas9n (Addgene #47929) as a template, and G(5′)ppp(5′)G RNA Cap Structure Analog (New England Biolabs, Ipswich, MA, USA). The transcribed RNAs were purified using a Sephadex column, followed by the phenol-chloroform extraction [47 (link)], prior to injection. sgRNA sequences can be found in the Table S1 .
8
Synthesis of Antisense RNA Probe for Whole-Mount in situ Hybridization
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To synthesize anti-sense RNA probe for whole-mount in situ hybridization, cDNA was PCR-amplified from total RNA extracted at 24 hpf embryos. The PCR fragments were cloned into the pGEM®-T Easy Vector (Promega, Madison, WI, United States). The cloned vectors were linearized with the NcoI restriction enzyme and then transcribed in vitro using SP6 RNA polymerase (Thermo Scientific, Waltham, MA, United States) with digoxigenin-labeled UTP (Roche, Basel, Switzerland). The following primers were used: odf3b (NCBI, acc. no. NM_199958) forward 5′-AGACGTCATGTCACCTGTG-3′ and odf3b reverse 5′-ATGACCTGCTTAATCTTCACCATCC-3′. Whole-mount in situ hybridization was performed as described previously (Thisse and Thisse, 2008 (link)).
9
Tol2-Mediated Zebrafish Line Generation
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Z48 and ZED zebrafish lines were generated through TOL2-mediated transgenesis93 (link). TOL2 cDNA was transcribed by Sp6 RNA polymerase (#EP0131, ThermoFisher Scientific) after Tol2-pCS2FA vector linearization with NotI restriction enzyme (#IVGN0016, Anza, Invitrogen, ThermoFisher Scientific). TOL2 mRNA was purified as previously described91 (link). One-cell stage embryos were injected with 1nL solution containing 25 ng/µL of transposase mRNA, 25 ng/µL of phenol/chloroform (#A931I500 and #C/4920/15, Fisher Chemical) purified plasmid (Z48 or ZED), and 0.05% phenol red (#P0290, Sigma-Aldrich).
10
Purification and In Vitro Transcription of SINV RNA
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The plasmids containing the SINV infectious clone were purified by the Plasmid Maxi Kit (Qiagen, Hilden, Germany), linearized by digestion with NotI (New England Biolabs, Ipswich, MA, USA), and purified by phenol-chloroform extraction. RNAs were then synthesized in an in vitro transcription system by incubating at 37°C for 1 h containing the linearized DNA template, NTP mix, dithiothreitol, RNaseOUT ribonuclease inhibitor, and SP6 RNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA). Following DNase I treatment, LiCl Precipitation Solution (Invitrogen) was added to purify the synthesized RNAs.
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