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.