The template for transcription of unmodified yeast tRNAiMet was generated by PCR, and RNA was synthesized using T7-MEGAshortscript (Invitrogen). RNA was purified by denaturing gel electrophoresis (8 M urea) in 1 x TBE buffer, eluted from the gel matrix and concentrated by ethanol precipitation. The template for in vitro transcription of mini Ty1 RNA (560 nt) was generated by PCR amplification of sequences corresponding to the RNA nt +1–560 from pBDG433 using a forward primer F-miniRNA containing an SP6 promoter sequence followed by 5′ Ty1 RNA sequence and a reverse primer R-miniRNA (Supplementary Table S2). DNA templates for PAL1, PAL2 and ∆S1a mini Ty1 RNA mutants were obtained using forward primers introducing desired mutations and primer R-mini RNA. Templates for the PAL3, SL4 and IL3 mutants were generated by PCR-driven overlap extension. All transcripts were synthesized using SP6-MEGAscript (Invitrogen) and purified using Direct-zol RNA MiniPrep Kit (Zymo Research). The quality of transcripts was monitored by high-resolution agarose-gel electrophoresis in the presence of formaldehyde. Mini Ty1RNA and Ty1 RNA mutants were 3′-end labeled with [α-32P] pCp using T4 RNA ligase (Fermentas) and purified on NucAway Spin Columns (Invitrogen Ambion). Purified RNAs were stored at −20°C.
Megascript sp6
Manufactured by Thermo Fisher Scientific
The Megascript SP6 is a laboratory equipment that is used for in vitro transcription. It generates high-quality SP6 RNA transcripts from DNA templates.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine rnaimax, by Thermo Fisher Scientific (6 mentions)
Luciferin substrate, by InvivoGen (2 mentions)
Thp 1 reporter cell lines, by InvivoGen (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Nucaway spin column, by Thermo Fisher Scientific (1 mentions)
T4 rna ligase, by Thermo Fisher Scientific (1 mentions)
Direct zol rna miniprep kit, by Zymo Research (1 mentions)
M7g 5 ppp 5 g cap analog, by Thermo Fisher Scientific (1 mentions)
Opti mem reduced serum medium, by Thermo Fisher Scientific (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
Rneasy minielute cleanup kit, by Qiagen (1 mentions)
Genejet plasmid miniprep kit, by Thermo Fisher Scientific (1 mentions)
Pgem t vector, by Promega (1 mentions)
Pcgn elk 1, by Addgene (1 mentions)
Nuclease free water, by Thermo Fisher Scientific (1 mentions)
7 protocols using megascript sp6
1
Synthesis and Purification of Yeast tRNA and Ty1 RNA
2
Recombinant SINV mRNA Transfection Assay
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The p5’dsMRE16ic-AaBec243–260-mCh-Flag and the p5’dsMRE16ic-mCh-Flag plasmids were linearized with AscI restriction enzyme and used as templates to generate capped SINV-AaBec243–260-mCh-Flag and SINV-mCh mRNA, respectively, using the SP6 MegaScript (Invitrogen) and m7 G(5′)ppp(5′)G Cap Analog (Ambion). BHK-21 cells in 6-well plates were transfected with the capped-SINV-AaBec243–260-mCh-Flag or capped-SINV-mCh-Flag mRNA using Lipofectamine LTX (Invitrogen) and Opti-MEM reduced serum medium (Gibco). Fluorescence was monitored daily for four days. The culture supernatants containing the virus particles were centrifuged at 13,600 x rcf for 5 min to remove cell debris. The supernatant was then aliquoted, stored at −80 °C, and used as seed stock. The recombinant SINV seed stock was further amplified using BHK-21 cells. Recombinant SINV strains were tested for genomic stability by passaging them at least seven times in BHK-21 cells while monitoring fluorescence. The virus stock was titrated using FFAs.
3
Synthetic Alu RNA Production Protocol
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D-PDB and DB were synthesized via reversible addition-fragmentation chain transfer polymerization as described previously (22 (link)). Alu DNA sequences were obtained from the GrCh37 (hg19) assembly. Synthetic Alu DNA with a SP6 promoter incorporated at the 5′ end was obtained from Integrated DNA Technologies (IDT DNA). Alu RNA was reverse transcribed using synthetic Alu DNA templates (IDT DNA) and MEGAscript SP6 (Invitrogen) in overnight reactions at 37°C. Reaction products were treated with Turbo DNase, precipitated with lithium chloride, and purified using the RNeasy MiniElute Cleanup Kit (Qiagen). Alu RNAs were not treated with phosphatases to remove 5′ phosphate groups. To quantitate yields, absorbance was determined at 260 nm. To ensure that the Alu RNAs were of the predicted size, gel electrophoresis was employed.
4
Alu-Mediated Transcriptional Regulation in Neurodegeneration
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Alu DNA sequences were from the GrCh37 (hg19) assembly. We designed unedited Alu DNA templates and changed A nucleotides edited in NCI HPC total vasculature but unedited in Alzheimer’s disease HPC total vasculature to G nucleotides as a mimic of A-to-I editing. Complete DNA sequences of unedited and edited MDM4 AluSg4 elements are shown in Supplementary Table 1 . A SP6 promoter was added to the 5′ end and synthetic double-stranded DNA templates were obtained from IDT. RNA transcription was performed using Megascript SP6 (Invitrogen) essentially as previously described.46 (link) THP-1 reporter cell lines (Invivogen) contain a stably integrated luciferase gene under the control of either an IFN-stimulated response element, ISRE or NF-κB response element. The human HMC3 microglial cell line was obtained from ATCC. THP-1 reporter or HMC3 cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (HyClone), glutamine, and penn/strep at 37°C in a humidified atmosphere of 5% CO2 in air. Transfections were performed using Lipofectamine RNAiMAX (Thermo Fisher Scientific).28 (link) Luciferase activity was determined after 24 h using luciferin substrate (Invivogen) and light emission measured with a TD20/20 luminometer. Gene expression measurements were performed essentially as previously described.25 (link),26 (link)
5
Plasmid Construction and RNA Probe Synthesis
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G-block gene fragments were ordered from IDT for ML_Fos3 (ML182032a), ML_Jun (ML1541120a), and ML_Etslx4 (ML282527a) coding sequences, then A-tailed and ligated each into Promega PGEM-T vector (A3600). Recombinant vectors into E. coli were transformed, and individual clones were selected for liquid culture and verified by colony PCR. Plasmids were purified using a Thermo Fisher scientific GeneJET Plasmid Miniprep kit (#K0503) and analyzed by Sanger sequencing to verify the orientation of the sequence (Supplementary Data 8 ). PCR primers specific to the bacterial promoter sequence were used to amplify the probe template from the minipreps, and DNA was purified using the Monarch PCR & DNA cleanup kit (#T1030L). DIG-labeled RNA probes (antisense and control sense) were transcribed using Invitrogen MEGAscript SP6 (#AM1330) or T7 (#AM1334) kit depending on the orientation of the sequence (Supplementary Fig. 8 ).
6
ACTA2-AS1 Transcription and Transfection
7
Alu DNA Editing and Reporter Assay
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