Protocol for massively parallel RNA assay combined with immunoprecipitation for high-throughput analysis of RNA-protein interactions in cells

RNA functions are largely mediated through interactions with RNA-binding proteins (RBPs), and defining the molecular principles underlying these interactions is essential for understanding RNA biology. Here, we present a massively parallel RNA assay combined with immunoprecipitation (MPRNA-IP) that enables high-throughput analysis of RNA-protein interactions in cells. We describe detailed steps for oligonucleotide design, cloning, transfection, sequencing, and computational analysis. Together, these procedures allow high-throughput interrogation of RNA sequences to identify sequence and structural elements that contribute to protein binding. For complete details on the use and execution of this protocol, please refer to Lee et al. 1