m 7 gpppg cap analog  (New England Biolabs)

Journal: iScience

Article Title: PABPC1 mediates degradation of C9orf72 -FTLD/ALS GGGGCC repeat RNA

doi: 10.1016/j.isci.2024.109303

Figure Lengend Snippet:

Article Snippet: In this step, to attach a 5′ cap to RNA, 10 mM 3′- O -Me-m 7 GpppG anti-reverse cap analog (ARCA) (New England Biolabs) was added, and then incubated at 37°C for 3 h. To remove plasmid DNA, the reaction mixture was treated with RQ1 RNase-free DNase (Promega) at 37°C for 15 min. Next, polyadenylation of the synthesized RNA was performed in reaction solution containing 1 mM ATP (New England Biolabs), E. coli Poly (A) Polymerase (New England Biolabs), and 4,000 U/mL Murine RNase inhibitor (New England Biolabs) at 37°C for 30 min. Then, 5′ cap- and 3′ poly (A)-attached synthesized RNA was purified using RNA Clean & Concentrator-25 kit (Zymo Research).

Techniques: FLAG-tag, Virus, Recombinant, Magnetic Beads, Plasmid Preparation, Protease Inhibitor, Blocking Assay, Reverse Transcription, Silver Staining, In Situ, Expressing, Construct, Software, Molecular Weight, Laser-Scanning Microscopy, Real-time Polymerase Chain Reaction