Protocol for measuring endocrine-disruptive effects on transcriptional bursting using single-molecule imaging in human breast cancer cells.

Transcriptional bursts regulate gene expression by altering burst size or burst frequency. Here, we present a protocol to analyze estrogen-responsive transcriptional bursting of the TFF1 gene in human breast cancer cell lines by integrating fixed-cell single-molecule RNA fluorescence in situ hybridization (smFISH) and live-cell single-molecule imaging. We describe cell seeding, ligand dose-response treatment, and smFISH hybridization and imaging to quantify transcription sites. We then detail live-cell imaging of a TFF1-MS2 reporter and integration of both datasets to resolve how endocrine disruptors modulate bursting dynamics. For complete details on the use and execution of this protocol, please refer to Day et al.1.