Identification of MEX3D Substrate RNAs in the Testis using eCLIP-seq
Identification of MEX3D Substrate RNAs in the Testis using eCLIP-seq
Testicular tissue from one side of the mouse was used per experiment. After removing the capsule, seminiferous tubules were lightly minced and dispersed in PBS. The sample was crosslinked on ice (254 nm, 400 Mj/CM², three times). After limited RNase I digestion, MEX3D-bound RNAs were dephosphorylated and ligated with a 3' linker. Protein-RNA complexes were separated by NativePAGE™ Bis-Tris Mini Protein Gels (Thermo, BN1001BOX) and transferred to a nitrocellulose membrane. The region above 75 kDa corresponding to the MEX3D-specific band was excised, and RNA fragments were recovered.  These RNA fragments were reverse transcribed into cDNA and ligated with a 5' linker. After qPCR quantification, the cDNA was amplified using VAHTS HiFi Amplification Mix (Vazyme, N616). 