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

Take a freshly harvested mouse testis in a chilled buffer. Remove the tunica albuginea, the fibrous tissue layer.

Triturate the testis to release seminiferous tubules containing germ and Sertoli cells.

Add buffer and subject the tubules to UV  radiation for irreversible crosslinking of RNA with associated proteins, preserving interactions.

Centrifuge and discard the supernatant.

Resuspend the tubules in a lysis buffer containing RNase and protease inhibitors.

Sonicate the tissue, releasing protein-RNA complexes and other intracellular components. The RNase and protease inhibitors inactivate endogenous RNases and proteases.

Add DNase to degrade DNA. Introduce diluted RNase for partial RNA digestion.

Centrifuge and transfer the supernatant containing RNA-protein complexes to an antibody-coated magnetic bead solution with high specificity to the target RNA-binding protein.

Apply a magnetic field to separate the bead-bound protein-RNA complexes. Discard the supernatant.

Wash with buffer. Magnetically separate the protein-RNA complexes and use them for further RNA-protein interaction analysis.

To begin this procedure, harvest about 100 milligrams of testes from mice of appropriate age for each immunoprecipitation experiment. Place the tissues in ice-cold PBS. Using a pair of fine-tip tip tweezers, gently remove the tunica albuginea. Add 3 milliliters of ice-cold PBS to a tissue grinder, and use a loose glass pestle to triturate the tissue by mild mechanical force. Next, transfer the tissue suspension to a cell culture dish, and add ice-cold PBS up to 6 milliliters. Shake the plate quickly so that liquid covers the bottom of the dish evenly.

Cross-link the suspension on ice three times with 400 millijoules per centimeter squared at 254 nanometers. Make sure to mix the suspension between each irradiation, then, collect the suspension in a 15-milliliter conical tube, and centrifuge at 1,200 times g and at 4 degrees for 5 minutes. Remove the supernatant and resuspend the pellet in 1 milliliter of PBS. After this, transfer the suspension to a 1.5-milliliter centrifuge tube. Centrifuge at 1,000 times g and at 4 degrees Celsius for two minutes and discard the supernatant.

First, add 125 microliters of protein A magnetic beads per sample to a fresh centrifuge tube. Place the tube on the magnet to separate the beads from the solution. After 10 seconds, remove the supernatant, and wash the beads twice with 1 milliliter of ice-cold lysis buffer. Resuspend the beads in 100 microliters of cold lysis buffer with 10 micrograms of eCLIP antibody. Rotate the tubes at room temperature for 45 minutes, then, wash the beads twice with 1 milliliter of ice-cold lysis buffer.

To begin, resuspend the tissue pellets in 1 milliliter of cold lysis buffer containing 22 microliters of 50X EDTA-free protein inhibitor cocktail and 11 microliters of RNAse inhibitor. Keep lysing the samples on ice for 15 minutes. Sonicate each sample is outlined in the text protocol. Next, add 4 microliters of DNAse to each tube and mix well. Incubate at 37 degrees Celsius for 10 minutes while shaking at 1,200 RPM. After this, add 10 microliters of diluted RNAse and mix well. Incubate at 37 degrees Celsius for 5 minutes while shaking at 1,200 RPM.

Then, centrifuge at 15,000 times g and at 4 degrees Celsius for 20 minutes to clear the lysate. Carefully collect the supernatant. First, add 1 milliliter of the lysate to the prepared beads and rotate the samples at 4 degrees Celsius for either two hours or overnight. After this, collect the beads with the magnetic stand and discard the supernatant. Wash the beads twice with 900 microliters of high-salt buffer, and then wash the beads twice with 900 microliters of wash buffer.