Protocol to measure transcriptional bursting of endogenous genes using high-throughput RNA-FISH

We present a protocol to measure transcriptional bursting of endogenous human genes that is widely applicable to adherent cells. We describe the steps for nascent RNA fluorescence in situ hybridization (nscRNA-FISH) in a high-throughput imaging format. We then detail the procedures for automated microscopy and analysis to measure bursting behavior of genes in adherent cell lines. The frequency of bursting alleles in a population is used as a readout for bursting behavior. For complete details on the use and execution of this protocol, please refer to Sood et al. 1 • Assay to visualize bursting of native genes using fluorescence in situ hybridization • Guidance for optimizing high-throughput cell culture in a 384-well plate format • Steps for high-throughput nascent RNA fluorescence in situ hybridization (nscRNA-FISH) • Analysis pipeline to measure gene bursting behavior from nscRNA-FISH Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. We present a protocol to measure transcriptional bursting of endogenous human genes that is widely applicable to adherent cells. We describe the steps for nascent RNA fluorescence in situ hybridization (nscRNA-FISH) in a high-throughput imaging format. We then detail the procedures for automated microscopy and analysis to measure bursting behavior of genes in adherent cell lines. The frequency of bursting alleles in a population is used as a readout for bursting behavior.