fRIP

Formaldehyde RNA Immunoprecipitation (fRIP) Protocol

Cross-linking mouse testes

1. Cross-link mouse testes in 0.1% formaldehyde at room temperature for 10 minutes while rotating.
2. Halt cross-linking by slowly adding glycine to a final concentration of 125mM and incubate at room temperature for 5 minutes.
3. Spin testes for 5 minutes at 500 g and wash twice in 4ºC PBS.
4. Flash-freeze testes and store at -80ºC.

Cross-linking Magnetic Beads to Antibody

1. Take 100uL of Protein A magnetic Dynabeads per IP (60uL beads for IP and 40uL for preclear).
2. Remove the supernatant and wash beads twice in 3% BSA in PBST (PBS w/ 0.1% tween).
3. Resuspend beads in 500uL 3% BSA in PBST.
4. Set aside 200uL for preclear and store at 4ºC.
5. 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].
6. Add 2ug antibody and incubate at 4°C overnight while rotating.

Day 2-

1. Remove supernatant and wash 3x at room temperature for 5 minutes in 1mL 0.2M triethanolamine pH 8.2.
2. Add 1mL dimethylprimelimidate (DMP) in 0.2M triethanolamine pH 8.2 to beads (5.4mg DMP in 1mL 0.2M triethanolamine).
3. Incubate at room temperature for 30 minutes while rotating.
4. Discard supernatant and add 1mL 50mM Tris, pH 7.5, wash 15 minutes withmixing.
5. Wash 3x with 1mL PBST.
6. Resuspend in 200uL PBST and store at 4°C.

Tissue Lysis

1. Add 275uL lysis buffer (with 100U/mL RNase-OUT) to each testis sample.
2. Mechanically disrupt testes with a pipet followed by a 25G needle after tissue is broken up.
3. Incubate lysate at 4°C for 15 minutes on rotator.
4. Sonicate samples using Bioruptor (1 cycle, 30 second pulse on Medium).
5. Spin cells down at maximum speed for 15 minutes at 4°C.
6. Collect supernatant and dilute by adding equal volume of binding/wash buffer (with 100U/mL RNase-OUT).
7. Quantify protein using the Bradford assay and use between 1-2 mg protein per IP.

Preclear Lysate

1. Wash preclear beads twice with 3% BSA PBST for 15 minutes at 4°C.
2. Add the lysate to the preclear beads and incubate for 1 hour at 4°C while rotating.
3. Set aside 1% precleared lysate for western and 10% for fRIP Input (keep Input lysate at 4°C).
    

Bead-Antibody Preparation

1. 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.
2. Wash the beads-antibody complex 3x in PBST.
3. Block the beads-antibody complex in 3% BSA PBST for 30 minutes while rotating.
4. Wash the beads-antibody complex 3x quickly in PBST.
5. Transfer to low binding tubes for immunoprecipitation.
    

Immunoprecipitation (IP)

1. Add precleared lysate to the beads-antibody complex and incubate for 4 hours at 4°C while rotating.
2. Saved supernatant and stored at -80°C.
3. Wash 4x with 1mL binding/wash buffer for 10 minutes at 4°C (transfer sample to new tube before last wash).
4. After final wash, transfer sample to new low binding tube.
5. Save 10% for western and 90% for RNAisolation.
6. For western samples:
   1. Resuspend beads in 100uL lysis buffer and transfer to new tube.
   2. Add 40uL fresh elution buffer to the beads and incubate at 70°C for 30 minutes with frequent vortexing.
   3. Transfer eluted sample to new tube, add laemmli buffer, boil and freeze.

Release of RNA

1. Resuspend beads in 93uL PK buffer, 5% (5uL) Proteinase K, 40Units (1uL) RNase-OUT and 15mM (1.5uL) 1M DTT (100uL total volume).
2. 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).
3. Incubate samples for 1 hour at 42°C, then another hour at 55°C.
4. Add 1mL TRIzol, mix, let sit ~5 minutes.
5. Add 200uL chloroform followed by 15 seconds agitation.
6. Centrifuge 20 minutes at 4°C max speed.
7. Collect aqueous layer, add 1uL glycoblue and 750uL isopropanol.
8. Incubate overnight at -20°C.
9. Centrifuge 40 minutes at 4°C max speed.
10. Wash 3x in 75% cold ethanol, spin 15 minutes at 9500rpm between washes.
11. Spin extra 5 minutes and air dry pellet.
12. Resuspend pellet in 20uL RNase-free water.
     

Lysis Buffer

**Add 100Units/mL RNaseOUT fresh.

Binding/ Washing Buffer

**Add 100Units/mL RNaseOUT fresh.

Elution Buffer

PK Buffer