Each cDNA for H2AX-EGFP, H3.3-EGFP, and H4-EGFP was cloned from an ovary cDNA library using a pUC118 vector (Takara Bio Inc., Shiga, Japan; 3318) containing the egfp sequence. Each cDNA for H2AXR3A-EGFP, H3.3R2A-EGFP, H3.3R8A-EGFP, H4R3A-EGFP and H3.3K4M-EGFP was cloned from the pUC118 vector containing each cDNA for H2AX-EGFP, H3.3-EGFP and H4-EGFP using mutagenesis primers (Table S3 ). These cDNAs were subcloned into a pcDNA3.1-poly (A) 83 vector53 (link). In vitro transcription was performed using a MEGAscript T7 Transcription Kit (Thermo Fisher Scientific, Massachusetts, USA; AM1333, AM1334) and Cap Analog (m7G(5′)ppp(5′)G) (Thermo Fisher Scientific; AM8048).
M7g 5 ppp 5 g cap analog
Manufactured by Thermo Fisher Scientific
Sourced in United States
The M7G(5')ppp(5')G Cap Analog is a synthetic RNA cap analog that can be used in various applications related to in vitro transcription and RNA research. It replicates the structure of the 5' cap found on eukaryotic mRNA molecules and can be used to produce capped RNA transcripts.
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Lab products found in correlation
Rneasy mini kit, by Qiagen (2 mentions)
M7gtp agarose, by Jena Biosciences (2 mentions)
M7gtp agarose, by Thermo Fisher Scientific (2 mentions)
Puc118 vector, by Takara Bio (1 mentions)
Megascript t7 transcription kit, by Thermo Fisher Scientific (1 mentions)
Pseudouridine triphosphate, by TriLink (1 mentions)
E coli poly a polymerase, by New England Biolabs (1 mentions)
N6 methyl atp, by TriLink (1 mentions)
2 o methyl atp, by TriLink (1 mentions)
Megascript t7 kit, by Thermo Fisher Scientific (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Sp6 polymerase, by Roche (1 mentions)
Mmessage mmachine kit, by Thermo Fisher Scientific (1 mentions)
Megascript sp6, 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)
6 protocols using m7g 5 ppp 5 g cap analog
1
Cloning and Transcription of Histone Variants
2
In Vitro Transcription and Modification of RNA
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Unmodified and modified RNA molecules were transcribed in vitro using the MEGAscript T7 kit (Thermo Fisher Scientific). Modified nucleotides triphosphates (2′O methyl-ATP, N6 methyl-ATP, pseudouridine triphosphate) were purchased from TriLink Biotechnologies. In vitro transcription reactions were assembled according to recommendation from the kit manufacturer and incubated overnight (16 h) at 37°C. Co-transcriptional capping was performed by substituting part of the GTP in the reaction with cap analog m7G(5′)ppp(5′)G (Thermo Fisher Scientific). After transcription, capped transcripts were treated with 1 μL thermosensitive shrimp alkaline phosphatase (tSAP; Promega) for 15 min at 37°C. All capped and uncapped transcripts were then treated with DNase I for 15 min at 37°C and immediately purified using the RNeasy Mini Kit (Qiagen). Post-transcriptional polyadenylation was performed on purified transcripts by adding 1 μL E. coli poly(A) polymerase (5 U/μL; New England Biolabs) and 1 mM ATP and incubating at 37°C for 30 min. Polyadenylation reactions were stopped by purification with the RNeasy Mini Kit. All transcripts were checked for purity and integrity by UV-vis spectrophotometry and denaturing poly-acrylamide gel electrophoresis (PAGE).
3
Cap-binding Protein Interaction Assay
4
Synthesis of Fluorescent mRNA for Live Imaging
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Fluorescent mRNAs for live imaging were synthesized as described below. The plasmids pCS2-β actin and pCS2-β-actin-Δ3′UTR were linearized with XbaI and BssHII, respectively, and used as templates for in vitro synthesis of β-actin mRNA with full-length and truncated 3′UTR respectively. In vitro transcription was carried out using SP6 polymerase (Roche) in the presence of 0.264 mM m7G(5')ppp(5')G Cap analog (Ambion), 0.4 mM ATP, 0.4 mM CTP, 0.12 mM GTP, and 0.4 mM UTP. One quarter of the total UTP (0.1 mM) was conjugated with the fluorophore cyanine 3 (Cy3) (PerkinElmer), Cy5 (PerkinElmer) or Alexa 488 (Invitrogen) to render the synthetic mRNA fluorescent. Transcription reactions were incubated for 2 h at 37°C. The synthesized RNA was treated with DNAse I (Stratagene), cleaned using the RNeasy Mini Kit (Qiagen), precipitated with lithium chloride (from the mMessage mMachine Kit, Ambion), and resuspended in RNAse-free water.
EB1-GFP mRNA for expression in Xenopus RGCs was synthesized as described below. The plasmid pCS2-EB1-GFP was linearized with NotI-HF and used as template for in vitro transcription, which was conducted using SP6 RNA polymerase (Ambion, mMachine mMessage SP6 Kit) at 37°C for 2 hr. The products were DNase-treated, cleaned and lithium chloride-precipitated as described above.
EB1-GFP mRNA for expression in Xenopus RGCs was synthesized as described below. The plasmid pCS2-EB1-GFP was linearized with NotI-HF and used as template for in vitro transcription, which was conducted using SP6 RNA polymerase (Ambion, mMachine mMessage SP6 Kit) at 37°C for 2 hr. The products were DNase-treated, cleaned and lithium chloride-precipitated as described above.
5
Cap-binding Protein Interaction Assay
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To analyze the interaction of METTL3 to cap-binding protein complex, cells were lysed using NET2 buffer and total cell extracts were incubated with m7GTP-Agarose (Jena Bioscience, AC-155S) for 2 hours at 4°C. Then, the beads were washed for five times and suspended in SDS sample buffer. The eluted samples were analyzed by Western blot. Where indicated, 75 μM of m7G(5’)ppp(5’)G Cap Analog (Ambion, AM8048) was added into the sample and incubated with m7GTP-Agarose.
6
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.
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