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. For complete details on the use and execution of this protocol, please refer to Buonaiuto et al. 1 • Design isoform-specific probes targeting a nuclear intron-retaining lncRNA • Prepare hiPSC-derived cardiomyocytes and apply RNA-FISH to detect nuclear lncRNAs • Perform subsequent immunofluorescence to detect RNA-protein nuclear complexes Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.