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This method describes the immunoprecipitation of RNA binding proteins from mammalian cell lines followed by the isolation of the bound RNAs for analysis by qPCR or seq techniques. The use of M2 anti-FLAG agarose beads may be replaced by the use of specific antibodies in conjunction with Sepharose G/A-beads.
Materials and Reagents
1. Anti-Flag M2 Affinity gel (Sigma Aldrich, catalog number: A2220)
2. RNase inhibitor (Invitrogen, catalog number: N8080119 or Bioline, catalog number: BIO-65028)
3. Complete Protease Inhibitor Cocktail Tablets (Roche, catalog number: 05892970001)
4. RnaseZap (Ambion, catalog number: AM9780)
5. Trizol reagent (Invitrogen, catalog number: 15596-026)
6. General chemicals (Sigma Aldrich)
7. DNase I recombinant, RNase-free inc. buffer (Roche, catalog number: 04716728001)
8. BioScriptTM (Bioline, catalog number: BIO-27036)
9. Formaldehyde
Equipment
1. Sonicator (the model is not critical but preferably a devise with a probe ≤ 5 mm in diameter)
2. Tube Rotator
3. Shaker
4. Centrifuge
5. PCR machine
6. Heating block
Procedure
I. Preparation of beads and cells
1. Blocking of the beads:
Wash 40 μl Anti-Flag M2 Affinity Gel twice with 900 μl of pure H2O (cold); add 400 μl IP lysis buffer +1% BSA; incubate at 4 °C (on a rotating wheel) overnight.
Alternatively use 30 μl SepharoseG-Beads + specific antibody. The amount of antibody and beads to be used in the precoupling will need to be determined in preliminary experiments. Typically using 10 μg of Ig for couplin...