Protocol for evaluating RNA-protein associations in mammalian cells with RIP-seq and RIP-qPCR.

RNA-protein interactions drive gene regulation, subcellular organization, and noncoding RNA function. Here, we present a protocol for measuring RNA-protein associations in formaldehyde-crosslinked mammalian cells using RNA immunoprecipitation followed by sequencing (RIP-seq) and quantitative PCR (RIP-qPCR). We include steps and best practices for qualifying reagents, preparing cells, and processing and analyzing data, including a standardized set of steps to quantify signal over noise. This protocol is broadly applicable for the study of RNA-protein interactions in cells. For complete details on the use and execution of this protocol, please refer to Trotman et al.1.