Protocol for the simultaneous detection of nuclear long non-coding RNAs and proteins in human iPSC-derived cardiomyocytes

Nuclear-enriched long non-coding RNAs (lncRNAs) can regulate gene expression by interacting with specific RNA-binding proteins (RBPs). Here, we present a protocol to directly image these lncRNAs at the single-molecule level together with their protein interactors in human cardiomyocytes. We describe steps to perform RNA-fluorescence in situ hybridisation (FISH) of a nuclear intron-retaining lncRNA, followed by sequential protein immunofluorescence (IF), enabling simultaneous visualisation of specific RNA and protein targets. We also detail procedures for probe design, cell seeding, and fixation.