Protocol for detecting and characterizing endogenous cis-antisense RNA duplexes and the associated RNA-binding proteins

Here, we present a protocol for detecting endogenous cis-antisense RNA duplexes and identifying their associated RNA-binding proteins in mammalian cells. We describe steps for validating RNA duplex formation, including J2 antibody-based RNA immunoprecipitation, RNA pull-down, and RNase protection assays. We further detail procedures for identifying and validating duplex-associated RNA-binding proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS), RNA pull-down, RNA immunoprecipitation, and electrophoretic mobility shift assays. Together, this workflow enables systematic characterization of endogenous cis-antisense RNA duplexes and their interacting proteins. For complete details on the use and execution of this protocol, please refer to Jiang et al.¹.