RNA Immunoprecipitation (RIP) Protocol

1. Cell Harvesting

1.1. Grow cells of the tissue culture cell line of interest to confluency and treat cells as required for the experiment.
1.2. If a cross-linking step is required this will require optimization of the fixation time, 
1.3. Harvest cells by trypsinization and resuspended in PBS (e.g. 10x7 cells in 2ml PBS), freshly prepared nuclear isolation buffer (2 ml) and water (6 ml), keep on ice for 20 min (with frequent mixing). One or more negative controls should be maintained throughout the experiment, e.g. no antibody sample or immunoprecipitation from knockout cells or tissue, knockdown cells are not recommended for negative control experiments.

2. Nuclei isolation and nuclear pellets lysis

2.1. Pellet nuclei by centrifugation at 2,500 g for 15 min.
2.2. Resuspend nuclear pellet in freshly prepared RIP buffer (1 ml).Avoid contamination using RNase-free reagents such as RNase-free tips, tubes and reagent bottles; also use ultraPURE distilled, DNase-free, RNase-free water to prepare buffers and solutions.

3. Shearing of chromatin

3.1. Split resuspended nuclei into two fractions of 500 ml each (for Mock and IP).
3.2. Mechanically shearing using a dounce homogenizer with 15–20 strokes.
Different cell lines might require optimization of shearing conditions.
3.3. Pellet nuclear membrane and debris by centrifugation at 13,000 rpm for 10 min.
Freeze an aliquot of lysate in liquid nitrogen for reference RNA isolation.

4. RNA Immunoprecipitation

4.1. Add antibody to protein of interest (2 to 10 ug) to supernatant (6 mg-10 mg) and incubate for 2 hr (to overnight) at 4 ℃ with gentle rotation.
4.2. Add protein A/G beads (40 ul) and incubate for 1 hr at 4oC with gentle rotation.
The amount of antibody that is added and the incubation time might need to be optimised depending on the protein of interest and antibody. If an antibody is working in IP, this is a good indication that it will work in RIP.

5. Washing off unbound material

5.1. Pellet beads at 2,500 rpm for 30 s, remove supernatant, and resuspend beads in 500 ml RIP buffer. Stringent washing of protein A/G bead pellets is important and might need to be optimized.
5.2. Repeat for a total of three RIP washes, followed by one wash in PBS.
Freeze five percent of the beads for SDS PAGE analysis after the second wash (e.g. use 5 μl of bead slurry if you have 100 μl total bead slurry volume).

6. Purification of RNA that was bound to immunoprecipitated RBP

6.1. Isolate coprecipitated RNAs by resuspending beads in TRIzol RNA extraction reagent (1 ml) according to manufacturer's instructions (further information can be found in our RNA isolation protocol
6.2. Elute RNA with nuclease-free water (e.g. 20 μl).
Add approximately 15-25 μl (depending on yield) of either DEPC treated TE buffer or water to the RNA pellet. Eluted RNA can be stored at -80°C. 6.3. Protein isolated by the beads can be detected by western blot analysis (further information can be found in our Western blot protocol.
If a cross-linking step has been used (1.2), the cross-link should now be reversed. Check out the reverse cross-links section of our ChIP protocol for details.

7. Reverse transcription (RT) of RNA to cDNA and analysis

7.1. Reverse transcription of DNAse treated RNA according to manufacturer's instructions (further information on DNAse treatment and Reverse transcription can be found in our RNA isolation protocol.
7.2. If target is known use qPCR of cDNA; if target is not known create cDNA libraries, microarrays and sequencing can be used for analysis.
The control experiments should give no detectable products after PCR amplification, and high-throughput sequencing of these control libraries should return very few unique sequences.
Reagents
Nuclear isolation buffer RIP buffer
1.28 M sucrose
40 mM Tris-HCl pH 7.5
20 mM MgCl2
4% Triton X-100
150 mM KCl
25 mM Tris pH 7.4
5 mM EDTA
0.5 mM DTT
0.5% NP40
100 U/ml RNAase inhibitor SUPERASin (add fresh each time)
Protease inhibitors (add fresh each time)