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8 protocols using sp6 scribe standard rna ivt kit

1

Synthesis of Radiolabeled dsRNA Substrates

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Radiolabeled dsRNA was prepared as described previously (Iki et al. 2017 ). Briefly, 34/36-nt dsRNA was prepared from two oligonucleotides (Gene Design). The 34-nt strand was end-labeled by incubation with [γ-32P]-dATP and T4 PNK (Thermo Scientific), whereas 36-nt strand was end-phosphorylated without radiolabeling. Single-stranded RNAs were annealed as follows: Guide and passenger strand were mixed together with 5× annealing buffer (Tris–HCl [pH 7.6] 50 mM, KCl 100 mM, and MgCl2 5 mM) and incubated for 2 min at 96°C in a thermomixer (Eppendorf), at which point the machine was turned off and ssRNAs were left to anneal with decreasing temperature for 12 h. 98/100- and 510/512-nt dsRNA substrates were created by in vitro transcription of individual strands using the SP6-Scribe Standard RNA IVT kit (Cellscript) with [α-32P]-CTP, purified on mini Quick Spin RNA Columns (Roche) and extracted with phenol:chloroform:isoamyl alcohol (PCI), followed by ethanol precipitation. Annealing of single-stranded RNA to obtain 98/100- and 510/512-nt dsRNA was achieved as described for 34/36-nt dsRNA species. All three species of duplexes contain a 5′ blunt end and 2 nt 3′ overhang.
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2

Capped and Polyadenylated mRNA Synthesis

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Reporter mRNA for was transcribed with a SP6-Scribe Standard RNA IVT Kit (CellScript). Polyadenylated BTF3_Fluc mRNA was transcribed from pSP36T-5′UTR_BTF3-FLuc-A50 linearized with HpaI. mRNAs were capped using the ScriptCap m7G Capping System and the ScriptCap 2′-O-methyltransferase enzyme (CellScript) according to the manufacturer's manual.
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3

Radioactive GFP Fragment Synthesis

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A partial GFP fragment amplified by PCR using TI429 and TI431 (Supplemental Table S1) was cloned between SalI and BamHI sites in pSP64-poly(A) vector (Promega). Plasmid linearized by EcoRI was used as template for in vitro transcription with the SP6-scribe standard RNA IVT kit (Cellscript) in the presence of [α-32P]-CTP. The transcripts were capped with the ScriptCap m7G Capping System (Cellscript). The products were purified as described in “Preparation of double-stranded RNA.”
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4

Synthesis and Purification of Radiolabeled RNA

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Complementary single-stranded RNAs were synthesized by in vitro transcription using the SP6-scribe standard RNA IVT kit (Cellscript) in the presence of [α-32P]-CTP. Template DNAs for in vitro transcription were partial gfp fragments containing SP6 promoter. The template fragments were amplified by PCR with oligonucleotides listed in Supplemental Table S1 (TI584, TI585, TI586, TI587, TI591, and TI592) and pSP-Flag-GFP as PCR template, and extracted by gel-purification. After in vitro transcription, the reaction mixtures were passed through mini Quick spin RNA columns (Roche), and purified RNA products were extracted by PCI and precipitated by ethanol. An equal amount of complementary RNAs was mixed and annealed as described in the “Small RNAs.”
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5

In Vitro Synthesis of Reporter mRNAs

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RNA was transcribed by T7 RNA polymerase using a HiScribe T7 High Yield RNA Synthesis Kit (NEB). Spike-in Fluc RNA was transcribed from FLuc Control Template (NEB), spike-in GFP RNA was transcribed from pGFP-c3 linearized with EcoRI. DNA template for Nluc mRNA was obtained by PCR amplification of the pNL2.2 SLU7-NlucP with the primers (Forward 5ʹ-TAATACGACTCACTATAGGGATTACGAGATTGGCTTGGATTC-3ʹand Reverse 5ʹ-TGTTGTTAACTTGTTTATTGCAGCTTATAATG-3ʹ, T7-promoter is underlined). RNAs were polyadenylated using the A-Plus Poly(A) Polymerase Tailing Kit (CellScript) according to the manufacturer’s manual.
Reporter mRNAs for in vitro translation experiments were transcribed with a SP6-Scribe Standard RNA IVT Kit (CellScript). Polyadenylated YB-3_Fluc_YB-3 mRNA was transcribed from pSP36T-5ʹUTR_YB-3-FLuc-3ʹUTR_YB-3-A50 linearized with HpaI. Polyadenylated beta-globin_Fluc mRNA was transcribed from pSP36TLuc-A50 linearized with SmaI. mRNAs were capped using the ScriptCap m7G Capping System and the ScriptCap 2ʹ-O-Methyltransferase Enzyme (CellScript) according to the manufacturer’s manual.
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6

Capped and m6A-Containing mRNA Synthesis

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Rabbit beta-globin mRNA was transcribed by T7 RNA polymerase from pET28a-BetaGlob-bGlob linearized with EcoRI. Firefly luciferase mRNA with 5′ UTR from rabbit beta-globin mRNA was transcribed by SP6 RNA polymerase from pSP36TBetaGlobFLucA50 linearized with SmaI. The transcription was performed using a SP6-Scribe Standard RNA IVT Kit (CellScript, WI, USA). For co-transcriptional rabbit beta-globin mRNA capping, the GTP concentration in the reaction mixture was reduced to 0.2 mM, and the m7G(5′)ppp(5′)G RNA cap structure analog (NEB, Ipswich, MA, USA) was added to 3.8 mM. The capped mRNA transcript for in vitro translation was obtained using a ScriptCap™ m7G Capping System and ScriptCap 2′-O-Methyltransferase Enzyme (CellScript, Madison, WI, USA) according to the manufacturers’ recommendations.
To obtain m6A-containing mRNA for experiments on the formation of 48S complexes, ATP was completely replaced by m6ATP at the same concentration (4 mM) in the transcriptional mixture. For translation in a cell-free system, mRNA containing 50% m6A was used. To obtain such mRNA, a mixture of 2 mM ATP and 2 mM m6ATP was used for in vitro transcription. The quality of the obtained mRNAs was checked by electrophoresis in 6% polyacrylamide gel (PAGE) containing 7 M urea.
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7

In vitro dsRNA Processing Assay

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The template ssRNA was synthesized by in vitro transcription using SP6-scribe standard RNA IVT kit (Cellscript), and purified as described in “Preparation of dsRNA.” The ∼100-nt ssRNA fragment is identical to the sense gfp fragment used for dsRNA preparation. The ∼1000-nt ssRNA fragment contains sequence corresponding to A. thaliana TAS1C. HA-RDR1, HA-RDR2, HA-RDR6, or GFP-HA (control condition) was expressed in BYL by in vitro translation, followed by immunopurification using TR buffer-equilibrated 10 µL anti-HA magnet beads (PIERCE). After the washing step with TR buffer, the magnet beads were incubated at 25°C for 2 h in the 20 µL reaction solution containing 750 nM 100-nt or 300 nM 1000-nt in vitro transcript as template together with 1mM ATP, 1 mM CTP, 1 mM GTP, 0.1 mM UTP, [α-32P]-UTP, and 12 mM MgCl2. The mixture was used for “dsRNA processing reaction.”
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8

Synthesis and Injection of Capped mRNAs

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To synthesize mRNA for microinjection, RNA was transcribed from a linearized plasmid DNA template using the SP6-Scribe Standard RNA IVT Kit (CELLSCRIPT) and purified with the RNeasy Mini Kit (QIAGEN). The m 7 G cap structure was added to the purified RNA using the ScriptCap m 7 G Capping System (CELLSCRIPT). Capped mRNA was purified with the RNeasy Mini Kit. For sfGFP reporter mRNAs, the poly(A) tail was added using the A-Plus Poly(A) Polymerase Tailing Kit (CELLSCRIPT), and the mRNA was then purified with the RNeasy Mini Kit. Purified mRNAs were diluted with water to the following concentrations: sfGFP reporter mRNAs and PACE reporter library 50 ng/µl; MT-Znf598 mRNAs 100 ng/µl; AnsB 50 ng/µl; tandem ORF reporter mRNAs 100 ng/µl. GFP MO was injected as described previously (Mishima and Tomari, 2016) (link). Microinjection was performed using an IM300 Microinjector (NARISHIGE). Approximately 1,000 pl of solution was injected per embryo within 15 min after fertilization.
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