Protocol for cell cycle-resolved high-content imaging of transcription condensate dynamics in human cells

Transcription condensates are dynamic, phase-separated compartments that control gene activation. Here, we present a protocol for studying transcription condensate dynamics in human cells. We describe steps for staining nascent DNA and endogenous condensate markers. We then detail procedures for automated confocal microscopy and analysis. Our high-throughput approach infers condensate dynamics across large timescales, allowing quantification of condensate changes across cell-cycle transitions and phases without synchronization. For complete details on the use and execution of this protocol, please refer to Marmolejo et al.¹.