An Enhanced Crosslinking Immunoprecipitation Method for Efficient Identification of Protein-bound RNA in Mouse Testes

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Tissue Harvesting and UV Crosslinking

1. Euthanize 2 adult mice using carbon dioxide (CO₂) for 1-2 min or until breathing stops. Next, perform cervical dislocation on each mouse.
2. Harvest about 100 mg of testes from mice of appropriate age (one adult testis in this study) for each immunoprecipitation experiment, and place the tissues in ice-cold phosphate-buffered saline (PBS).
3. Remove the tunica albuginea gently with one pair of fine-tipped tweezers.
4. Add 3 mL of ice-cold PBS in a tissue grinder and triturate the tissue by mild mechanical force using a loose glass pestle (type A glass pestle).
   NOTE: The purpose of this step is not to lyse cells, but to pull apart the tissue. Preservation of cell viability and integrity is important.
5. Transfer the tissue suspension to a cell culture dish (10 cm in diameter) and add ice-cold PBS up to 6 mL.
6. Shake the plate quickly so that the liquid covers the bottom of the dish evenly. If the tissue is ground properly, evenly distributed seminiferous tubules will be visible, whereas the presence of tissue clumps indicates that tissue dispersion is suboptimal.
7. Crosslink the suspension three times with 400 mJ/cm² at 254 nm on ice. Mix suspension between each irradiation.
   NOTE: For each new experiment, crosslinking should be optimized.
8. Collect the suspension in a 15 mL conical tube and pellet at 1,200 x g for 5 min at 4 °C. Remove the supernatant, resuspend the pellet in 1 mL of PBS, and then transfer the suspension to a 1.5 mL centrifuge tube. Spin at 4 °C and 1,000 x g for 2 min, and remove supernatant.
9. At this point, immediately proceed with the rest of the protocol, or snap-freeze the pellets in liquid nitrogen and store them at -80 °C until use.

2. Beads Preparation

1. Add 125 µL of protein A magnetic beads per sample (pellet) to a fresh centrifuge tube.
   NOTE: Use protein G magnetic beads for mouse antibodies.
2. Place the tube on the magnet to separate the beads from the solution. After 10 s, remove the supernatant. Wash beads twice with 1 mL of ice-cold lysis buffer.
   NOTE: Subsequent separation of protein A magnetic beads follows this step. Lysis buffer composition is 50 mM Tris-HCl, pH 7.5; 100 mM NaCl; 1% NP-40; 0.1% SDS; 0.5% sodium deoxycholate; 1/50 ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail (add fresh).
3. Resuspend beads in 100 µL of cold lysis buffer with 10 µg eCLIP antibody. Rotate tubes at room temperature for 45 min.
   NOTE: The final antibody concentration for immunoprecipitation is 10 µg/mL. If the antibody concentration is unknown, the amount of antibody should be optimized.
4. Wash beads twice with 1 mL of ice-cold lysis buffer.

3. Tissue Lysis and Partial RNA Digestion

1. Resuspend tissue pellets in 1 mL of cold lysis buffer (add 22 µL of 50x (EDTA)-free protein inhibitor cocktail and 11 µL of RNase inhibitor to 1 mL of lysis buffer).
   1. Resuspend two UV-crosslinked pellets and two non-crosslinked pellets per group of experiments: UV-crosslinked-1 pellets for eCLIP library (UV-1); UV-crosslinked-2 pellets for eCLIP library (UV-2); non-crosslinked-1 pellets for eCLIP library as a control (non-UV); non-crosslinked-2 pellets for IgG IP to demonstrate the specificity of antibodies.
      NOTE: The ideal control for the eCLIP library of the UV-crosslinked wide-type testes is that of the UV-crosslinked knockout testes from mice of the same litter.
2. Keep lysing the samples on ice for 15 min (to prevent degradation of protein and RNA).
3. Sonicate in a digital sonicator at 10% amplitude for 5 min, at 30 s on/30 s off. Always place the sample on ice and clean the probe with nuclease-free water between each sample.
4. Add 4 µL of DNase to each tube, and mix well. Incubate for 10 min at 37 °C, shaking at 1,200 rpm.
5. Add 10 µL of diluted RNase I (4 U/µL RNase I in PBS), and mix well. Incubate for 5 min at 37 °C, shaking at 1,200 rpm.
6. Clear the lysate by centrifugation at 15,000 x g for 20 min (at 4 °C).
7. Carefully collect the supernatant. Leave 50 µL of lysate and discard the pellet with it.
8. Save input samples as RWB (run for western blot) and RRI (run for RNA isolation). Save 20 µL (2%) of UV-1, UV-2, non-UV, and IgG samples as inputs for RWB gel loading. Save 20 µL (2%) of UV-1 or UV-2 samples as inputs for RRI gel loading.

4. Immunoprecipitation

1. Add 1 mL of the lysate (from step 3.7) to the beads (prepared in section 2) and rotate the samples at 4 °C for 2 h or overnight.
2. Collect the beads with a magnetic stand and discard the supernatant. Wash the beads twice with 900 µL of high salt buffer (50 mM Tris-HCl, pH 7.5; 1 M NaCl; 1 mM EDTA; 1% NP-40; 0.1% SDS; 0.5% sodium deoxycholate), and then wash beads twice with 900 µL of wash buffer (20 mM Tris-HCl, pH 7.5; 10 mM MgCl2; 0.2% Tween-20).
   NOTE: For the IgG sample, pause the procedure here and store it on ice in a wash buffer.