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.