Biotin-Labelling of Immunoprecipitated RNA (v2) v1

RNA binding proteins (RBPs) regulate a diverse array of RNA processing steps by binding RNAs through sequence and structural elements. The development of crosslinking and immunoprecipitation (CLIP) enabled identification of RBP targets transcriptome-wide in a robust manner. During CLIP, it is often desired to visualize RBP:RNA complexes after denaturing SDS-PAGE electrophoresis and transfer to nitrocellulose membranes. However, standard methods used radiolabeling of RNA followed by autoradiography, introducing significant challenges for usability and scaling experiments to profile many RBPs. Here we describe an alternative approach to visualize RBP:RNA complexes using ligation of biotinylated nucleotides, followed by standard chemiluminescent imaging. This approach retains the advantages of visualization while decreasing handling complexity, enabling large-scale experiments to verify the presence and sizing of immunoprecipitated RBP:RNA complexes.