Formaldehyde RNA Immunoprecipitation (fRIP) Protocol
Cross-linking mouse testes
- Cross-link mouse testes in 0.1% formaldehyde at room temperature for 10 minutes while rotating.
- Halt cross-linking by slowly adding glycine to a final concentration of 125mM and incubate at room temperature for 5 minutes.
- Spin testes for 5 minutes at 500 g and wash twice in 4ºC PBS.
- Flash-freeze testes and store at -80ºC.
Cross-linking Magnetic Beads to Antibody
- Take 100uL of Protein A magnetic Dynabeads per IP (60uL beads for IP and 40uL for preclear).
- Remove the supernatant and wash beads twice in 3% BSA in PBST (PBS w/ 0.1% tween).
- Resuspend beads in 500uL 3% BSA in PBST.
- Set aside 200uL for preclear and store at 4ºC.
- Resuspend the remaining beads in 200uL lysis buffer [40uL 1M Tris (20mM final)+ 54uL 5M NaCl (135mM final) +200uL 100% glycerol (10% final) +200uL 10% NP40 (1% final) +40uL 5M EDTA (5mM final) + 1466 water].
- Add 2ug antibody and incubate at 4°C overnight while rotating.
Day 2-
- Remove supernatant and wash 3x at room temperature for 5 minutes in 1mL 0.2M triethanolamine pH 8.2.
- Add 1mL dimethylprimelimidate (DMP) in 0.2M triethanolamine pH 8.2 to beads (5.4mg DMP in 1mL 0.2M triethanolamine).
- Incubate at room temperature for 30 minutes while rotating.
- Discard supernatant and add 1mL 50mM Tris, pH 7.5, wash 15 minutes withmixing.
- Wash 3x with 1mL PBST.
- Resuspend in 200uL PBST and store at 4°C.
Tissue Lysis
- Add 275uL lysis buffer (with 100U/mL RNase-OUT) to each testis sample.
- Mechanically disrupt testes with a pipet followed by a 25G needle after tissue is broken up.
- Incubate lysate at 4°C for 15 minutes on rotator.
- Sonicate samples using Bioruptor (1 cycle, 30 second pulse on Medium).
- Spin cells down at maximum speed for 15 minutes at 4°C.
- Collect supernatant and dilute by adding equal volume of binding/wash buffer (with 100U/mL RNase-OUT).
- Quantify protein using the Bradford assay and use between 1-2 mg protein per IP.
Preclear Lysate
- Wash preclear beads twice with 3% BSA PBST for 15 minutes at 4°C.
- Add the lysate to the preclear beads and incubate for 1 hour at 4°C while rotating.
- Set aside 1% precleared lysate for western and 10% for fRIP Input (keep Input lysate at 4°C).
Bead-Antibody Preparation
- Pre-elute the cross-linked beads-antibody complex to remove any noncross-linked antibody by washing quickly twice with 1mL 100mM glycine pH 2.5.
- Wash the beads-antibody complex 3x in PBST.
- Block the beads-antibody complex in 3% BSA PBST for 30 minutes while rotating.
- Wash the beads-antibody complex 3x quickly in PBST.
- Transfer to low binding tubes for immunoprecipitation.
Immunoprecipitation (IP)
- Add precleared lysate to the beads-antibody complex and incubate for 4 hours at 4°C while rotating.
- Saved supernatant and stored at -80°C.
- Wash 4x with 1mL binding/wash buffer for 10 minutes at 4°C (transfer sample to new tube before last wash).
- After final wash, transfer sample to new low binding tube.
- Save 10% for western and 90% for RNAisolation.
- For western samples:
- Resuspend beads in 100uL lysis buffer and transfer to new tube.
- Add 40uL fresh elution buffer to the beads and incubate at 70°C for 30 minutes with frequent vortexing.
- Transfer eluted sample to new tube, add laemmli buffer, boil and freeze.
Release of RNA
- Resuspend beads in 93uL PK buffer, 5% (5uL) Proteinase K, 40Units (1uL) RNase-OUT and 15mM (1.5uL) 1M DTT (100uL total volume).
- To Input lysate samples, add 43uL PK buffer, 5% (5uL) Proteinase K, 40Units (1uL) RNase- OUT and 15mM (1.5uL) 1M DTT (100uL total volume).
- Incubate samples for 1 hour at 42°C, then another hour at 55°C.
- Add 1mL TRIzol, mix, let sit ~5 minutes.
- Add 200uL chloroform followed by 15 seconds agitation.
- Centrifuge 20 minutes at 4°C max speed.
- Collect aqueous layer, add 1uL glycoblue and 750uL isopropanol.
- Incubate overnight at -20°C.
- Centrifuge 40 minutes at 4°C max speed.
- Wash 3x in 75% cold ethanol, spin 15 minutes at 9500rpm between washes.
- Spin extra 5 minutes and air dry pellet.
- Resuspend pellet in 20uL RNase-free water.
Lysis Buffer
| Stock Reagent | Amount for 1mL | Final concentration |
| 1M Tris-HCl pH 8 | 50uL | 50mM |
| 2M KCl | 75uL | 150mM |
| 10% SDS | 10uL | 0.1% |
| 10% Triton X-100 | 100uL | 1% |
| 0.5M EDTA | 10uL | 5mM |
| 10% Sodium Deoxycholate | 50uL | 0.5% |
| 1M DTT | 0.5uL | 0.5mM |
| Complete protease Inhibitor (CPI) 7x | 150uL | 1x |
| Nano pure water | 555uL | n/a |
**Add 100Units/mL RNaseOUT fresh.
Binding/ Washing Buffer
| Stock Reagent | Amount for 1mL | Final concentration |
| 1M Tris-HCl pH 7.5 | 25uL | 25mM |
| 2M KCL | 75uL | 150mM |
| 0.5M EDTA | 10uL | 5mM |
| 10% NP-40 | 50uL | 0.5% |
| 1M DTT | 0.5uL | 0.5mM |
| Complete protease Inhibitor (CPI) 7x | 150uL | 1x |
| Nano pure water | 690uL | n/a |
**Add 100Units/mL RNaseOUT fresh.
Elution Buffer
| Stock reagent | Amount for 1mL | Final concentration |
| 10% SDS | 100ul | 1% |
| 0.5M EDTA | 20uL | 10mM |
| 1M Tris-HCL, pH 8.0 | 50uL | 50mM |
| 100mM PMSF | 10uL | 1mM |
| 7x CPI | 150uL | 1x |
| Nano pure water | 670uL | n/a |
PK Buffer
| Stock reagent | Amount for 25mL | Final concentration |
| 1M Tris pH 7.0 | 250uL | 10mM |
| 5M NaCl | 500uL | 100mM |
| 0.5M EDTA | 50uL | 1mM |
| 10% SDS | 1250uL | 0.5% |
| Nano pure water | 22950uL | n/a |