Sp6 scribe standard rna ivt kit

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.”

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.

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.”