Protocol for using NudC in decapping RNA containing non-canonical initiating nucleotides (NEB #M0607)
- Assemble the following reaction on ice:
COMPONENT 20 μl REACTION E. coli total RNA* up to 2 µg NEBuffer r3.1 (10X) 2 µl 100 mM DTT 1 µl NudC pyrophosphatase (10 µM) 0.5 – 1 µl Nuclease-free Water to 20 µl
- Incubate at 37°C for 30 min.
- NudC reactions can be stopped by addition of 10 mM EDTA or heating the reaction at 65°C for 10 min. If neither treatment is desirable, NudC, along with reaction components can be removed from the RNA using RNA purification methods such as column purification (NEB recommends the NEB Monarch® RNA Cleanup Kits) or phenol/chloroform extraction followed by ethanol precipitation.
* E. coli total RNA is used in this protocol. For applications in eukaryotic RNA, where non-canonical cap structures are to be distinguished from G-capped RNA and 5′ monophosphorylated RNA in a 5′ ligation-based workflow, NEB recommends pre-treating the RNA sample with yDcpS (NEB #M0463), followed by Quick CIP (NEB #M0525) and a RNA purification step to turn G-capped RNA and existing 5’ phosphorylated RNA to 5′ hydroxyl RNA, which is not available for ligation.