So you’ve been following RIP and CLIP over the last couple of years and you’ve seen what researchers can do with these methods. Now it’s time to roll up the sleeves and put the pipette in action.
The kinds folks over at Abcam have some nice protocol guides to help you get fastracked to RIP and CLIP.
This protocol had been adapted Khalila et al. 2009, Hendrickson et al. 2009 and 2008, and Rinn et al. 2007 and provides useful guidance and buffer recipes to get you up and running with RIP.
This detailed protocol adapted from Konig et al. J. Vis. Exp. 2011 takes you through every step of the iCLIP protocol including:
- UV cross-linking of tissue culture cells
- Bead preparation for later immunoprecipitation (IP)
- Cell lysis and partial RNA digestion
- IP and dephosphorylation of RNA 3’ends
- Linker ligationto RNA 3′ ends and RNA 5′ end labeling
- SDS-PAGE and membrane transfer
- RNA isolation
- Reverse transcription (RT)
- Gel purification of cDNA
- Ligation of primer to the 5′ end of the cDNA
- PCR amplification