Plasmids and oligonucleotides are listed in Supplementary Table S1. RNA and DNA oligonucleotides were obtained from Integrated DNA Technologies (IDT) (Corvalville, IA, USA) and purified by denaturing polyacrylamide gel electrophoresis (PAGE) before use. The 3′deoxyG13 RNA was obtained from Thermo Fisher Scientific Biosciences Inc. [γ-32P]ATP, [α-32P]CTP and [α-32P]GTP were from PerkinElmer Life Sciences; rNTPs were from Promega (Madison, WI, USA).
Rntps
Manufactured by Promega
Sourced in United States
RNTPs are a set of ribonucleotides, which are the building blocks for RNA synthesis. These essential components are required for in vitro RNA production and various molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
α 32p gtp, by PerkinElmer (2 mentions)
γ 32p atp, by PerkinElmer (2 mentions)
α 32p ctp, by PerkinElmer (2 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (2 mentions)
Acrylamide bisacrylamide, by Bio-Rad (2 mentions)
Hybond n membrane, by GE Healthcare (2 mentions)
Taq dna polymerase, by New England Biolabs (2 mentions)
T4 dna ligase, by New England Biolabs (2 mentions)
Agarose, by Thermo Fisher Scientific (2 mentions)
32p ntps, by PerkinElmer (2 mentions)
T4 polynucleotide kinase, by New England Biolabs (2 mentions)
Hybond, by Cytiva (2 mentions)
Megascript rnai kit, by Thermo Fisher Scientific (1 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
T7 rna polymerase, by Thermo Fisher Scientific (1 mentions)
Pegfp c1, by Takara Bio (1 mentions)
Prl cmv, by Promega (1 mentions)
α 32p rctp, by PerkinElmer (1 mentions)
Oligonucleotide, by Integrated DNA Technologies (1 mentions)
Rnase 1, by Thermo Fisher Scientific (1 mentions)
10 protocols using rntps
1
Radiolabeled RNA Oligonucleotide Synthesis
2
In Vitro Transcription of dsRNA
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dsRNA molecules against either FFLuc or RLuc were produced using a T7 RNA polymerase in vitro transcription kit (Megascript RNAi kit, Ambion, Foster City, CA, USA) using a PCR product template flanked by T7 RNA polymerase promoter sequences. pIZ-Fluc [45 (link)] and pRL-CMV (Promega, Madison, WI, USA) were used as a template for the amplification of dsRNAs targeting FFLuc and RLuc, respectively. An eGFP-derived dsRNA was taken as a control. This sequence was obtained from a gel-purified PCR product using peGFP-C1 (Clontech, Mountain View, CA, USA) as a template. Primer sequences can be found in Table S1 .
Internally radio-labelled dsRNAs were prepared by combining 5 µL 114 nt eGFP PCR product with T7 polymerase sites, 4 µL 5× Transcription buffer (Ambion), 2 µL DTT (0.1 M, Invitrogen, Carlsbad, CA, USA), 1 µL rNTPs (10 mM each ATP, GTP and UTP with 0.1 mM CTP) (Promega), 3 µL α-32P rCTP (Perkin Elmer, Waltham, MA, USA), 1 µL T7 RNA polymerase (Ambion), 1 µL RNase inhibitor (Invitrogen), and 3 µL RNase/DNase free H2O. The reaction was incubated for 1 to 3 h at 37 °C before heating to 65 °C for 5 min and allowing it to gradually cool to room temperature. Following this, 2 µL of DNase I and 1 µL of RNase A were added and the reaction was incubated for a further 30 min at 37 °C. The dsRNA could then be purified by running on an 8% native acrylamide gel.
Internally radio-labelled dsRNAs were prepared by combining 5 µL 114 nt eGFP PCR product with T7 polymerase sites, 4 µL 5× Transcription buffer (Ambion), 2 µL DTT (0.1 M, Invitrogen, Carlsbad, CA, USA), 1 µL rNTPs (10 mM each ATP, GTP and UTP with 0.1 mM CTP) (Promega), 3 µL α-32P rCTP (Perkin Elmer, Waltham, MA, USA), 1 µL T7 RNA polymerase (Ambion), 1 µL RNase inhibitor (Invitrogen), and 3 µL RNase/DNase free H2O. The reaction was incubated for 1 to 3 h at 37 °C before heating to 65 °C for 5 min and allowing it to gradually cool to room temperature. Following this, 2 µL of DNase I and 1 µL of RNase A were added and the reaction was incubated for a further 30 min at 37 °C. The dsRNA could then be purified by running on an 8% native acrylamide gel.
3
Detailed Nucleic Acid Preparation Protocol
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Plasmids, Oligonucleotides, cell strains are listed in Supplementary Table S1 . Oligonucleotides were obtained from Integrated DNA Technologies (Corvalville, IA, USA) and RNA Oligonucleotides and NET-seq cDNA library primers were purified with polyacrylamide gel electrophoresis (PAGE). [α-32P]GTP, [α-32P]CTP and [γ-32P]ATP were from PerkinElmer Life Sciences; rNTPs were from Promega (Madison, WI, USA).
4
Evaluating pri-miRNA Secondary Structure
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DNA products of the sequences carrying the G‐ or A‐allele controlled by the T7 promoter were obtained by PCR. In vitro transcription was performed following the manufacturer’s protocol of the T7 RNA polymerase (T7 polymerase Roche Cat# 10881767001). Briefly, the same amount of DNA was incubated with 1 mM rNTPs (Promega Cat#P113B), 20 U RNase inhibitor (Promega Cat# N261B) 2 h at 37°C and RNA purified with DirectzolT RNA Miniprep (ZymoResearch # ZYR2052). After, in vitro annealing of the secondary structure was done by incubating the same amount of RNA for each sample in duplicate at 70°C for 2 min in 10 mM Tris–HCl pH 7.5, 100 nM NaCl, 1 mM EDTA following a 15 min cool down of the samples at room temperature. For RNA cleavage of the secondary structure obtained, one of the duplicates produced was used as control of the digestion (no enzyme). Then, 5 U of RNaseI (Thermo Scientific Cat# EN0601) were added for the digestion in the Enzyme samples for 30 min at 37°C and inactivated at 100°C for 20 min. RNA was extracted using DirectzolT RNA Miniprep (ZymoResearch # ZYR2052). Finally, to evaluate the pri‐miRNA, cDNA was produced using the same amount of RNaseI‐digested RNAs for all samples using High‐Capacity cDNA RT kit (Applied Biosystems Cat# 4368813) and RT–qPCR performed using SYBR Green (Promega).
5
Synthesis and Purification of Irf7 mRNA
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pBluescript II SK (+) Irf7 5’-UTR 1-215 was incubated with T7 polymerase (Enzynomics) and mixture (RNase-free water, rNTPs (Promega), 100 mM DTT (Invitrogen), recombinant RNasin (Promega), 10× T7 polymerase (Enzynomics) buffer) at 37℃ for 1 h to generate mRNA. RQ1 DNase (Promega) was added to the mixture and the incubated was continued at 37℃ for 30 min to remove the templates. The newly generated mRNAs were purified by phenol-chloroform precipitation (RNase-free water 80 μl, phenol:chloroform:isoamyl alcohol 25:24:1, saturated with 10 mM Tris, pH 8.0, 1 mM EDTA (Sigma) 100 μl, 100% ethanol (Sigma) 275 μl, 5 μM NH4Ac (Sigma) 10 μl, 75% ethanol 100 μl).
6
T4 Polynucleotide Kinase and Taq DNA Polymerase Protocol
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T4 polynucleotide kinase and Taq DNA polymerase were from New England Biolabs, Pfu-Turbo DNA polymerase was from Stratagene, T4 DNA ligase was from New England Biolabs. DNA oligonucleotides were custom synthesized by Sigma Aldrich or Eurofins MWG/Operon. The complete list of DNA oligonucleotides used in this study is shown in Table S2 . DNA sequencing was performed by GATC biotech. Acrylamide-bisacrylamide and other electrophoresis reagents were from BioRad. Agarose was from Invitrogen. Hybond-N+ membranes and hybridization buffer used for Northern blot analysis were from GE Healthcare and from Applied Biosystems-Ambion, respectively. The rNTPs were from Promega and the 32P-NTPs were from PerkinElmer or Hartmann Analytic. 32P-labeled nucleic acids were detected by phosphorimaging using ImageQuant software.
7
Molecular Biology Reagents and Techniques
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T4 polynucleotide kinase, T4 DNA ligase, Taq DNA polymerase, and restriction enzymes were purchased from New England Biolabs. Pfu-Turbo DNA polymerase was from Stratagene. Sigma-saturated RNA polymerase from E. coli was obtained from Epicentre. DNA oligonucleotides were custom-synthesized by Sigma-Aldrich, Eurogentec, or Eurofins MWG/Operon. Most chemicals were purchased from Sigma-Aldrich. Acrylamide–bisacrylamide and other electrophoresis reagents were from Bio-Rad and Amresco. Agarose was from Invitrogen. Hybond-N+ membranes and hybridization buffer used for Northern blot analysis were from GE Healthcare and Applied Biosystems-Ambion, respectively. The rNTPs were from Promega, and the 32P-NTPs were from PerkinElmer or Hartmann Analytic. 32P-labeled nucleic acids were detected by phosphorimaging using ImageQuant software.
8
Quantifying Unconventional Splicing via In Vitro Transcription
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In vitro transcription was performed based as reported previously (39 (link)). Briefly, 5 μg linearized plasmid DNA (pSI-lplE4-Ren-lplE42 or pSI-lplE4-LacZ-lplE42) was incubated with nuclear fraction from HVSMCs cultured with either Ad-null or Ad-XBP1s, NEB 10× transcription buffer, and 100 mM rNTPs (Promega) at 37°C for 2 h and then digested with DNase I at 37 °C for 30 min. RNA extraction was performed using Phenol/Chloroform (pH 4.5), followed by RT-PCR with primer set of 5’-tac gac tca cta tag gct agc-3’ versus 5’-gat ccg ctc tag gtt taa acg-3’ to amplify a 80 bp fragment derived from unconventional splicing or recombinate between the two loxP-like elements.
9
Oligonucleotide Purification and Radiolabeling
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DNA and RNA oligonucleotides were purchased from Integrated DNA Technologies (IDT;
Coralville, IA) and purified by 15% denaturing polyacrylamide gel electrophoresis (PAGE) before use. [g-32 P]ATP, [a-32 P]CTP and [a-32 P]GTP were obtained from PerkinElmer Life Sciences; rNTPs, from Promega (Madison, WI, USA); and 3′deoxy ATP (3′dATP) and CMPCPP, from Jena Bioscience. H2O2 30% (w/w) solution and standard buffers and reagents were from Sigma-Aldrich or Thermo-Fisher.
Coralville, IA) and purified by 15% denaturing polyacrylamide gel electrophoresis (PAGE) before use. [g-32 P]ATP, [a-32 P]CTP and [a-32 P]GTP were obtained from PerkinElmer Life Sciences; rNTPs, from Promega (Madison, WI, USA); and 3′deoxy ATP (3′dATP) and CMPCPP, from Jena Bioscience. H2O2 30% (w/w) solution and standard buffers and reagents were from Sigma-Aldrich or Thermo-Fisher.
10
Nonradioactive in situ Hybridization Protocol
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Nonradioactive in situ hybridization was performed essentially as previously described (Carcagno et al., 2014) . Briefly, sections were fixed 15 min with paraformaldehyde (PFA) 4% in PBS and washed with PBS-DEPC. Tissue was treated with proteinase K (3 mg/mL, 3 min), followed by PFA 4% for 10 min and PBS washes. Slides were incubated in triethanolamine-acetic anhydrate pH 8.0 for 10 min, permeabilized with Triton X-100 1% in PBS for 30 min, and washed with PBS. Sections were incubated for 2 h with hybridization solution (50% formamide, 53 SSC, 53 Denhardt solution, 250 mg/mL yeast tRNA). Digoxigeninlabeled RNA probes were generated by in vitro transcription using T7 RNA polymerase (Promega), digoxigenin-UTP (Roche), rNTPs (Promega), and polymerase chain reaction (PCR)-amplified products or linearized plasmids as templates. RNA probes used were mSim1 (Zhang et al., 2008 ), mNkx2.2 (Briscoe et al., 1999) , mUncx (Mansouri et al., 2000) , and mvGluT2 (Slc17a6) (Lanuza et al., 2004) .
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