Protocol for massively parallel RNA assay coupled with immunoprecipitation (MPRNA-IP)
RNA mediates many cellular functions through interactions with RNA-binding proteins (RBPs), and defining the molecular principles underlying these interactions is essential for understanding RNA biology. Here, we describe a detailed protocol for MPRNA-IP, a Massively Parallel RNA assay coupled with ImmunoPrecipitation that enables high-throughput dissection of RNA–protein interactions in cells. This protocol provides a complete experimental and computational workflow, including the design of RNA tiles, large-scale oligonucleotide cloning, mammalian cell transfection, immunoprecipitation of RNA–protein complexes, sequencing library preparation, and downstream data analysis. As an example application, we synthesized a pool of oligonucleotides tiling six lncRNAs and studied PUM2-binding motifs. Together, these procedures allow high-throughput interrogation of RNA sequences to detect sequence and structural elements that contribute to protein binding. For further information on the development and applications of MPRNA-IP, see Lee et al., Nucleic Acids Research, 2024 (https://doi.org/10.1093/nar/gkae334). Overall design: Overexpress a pool of oligonucleotides and perform immunoprecipitation in three biological replicates