Droplet formation assay for investigating phase-separation mechanisms of RNA Pol II transcription and CTCF functioning.

Clustering of RNA polymerase II carboxy-terminal domain (CTD) and CTCF DNA-binding domains (DBDs) have been viewed as emerging mechanisms of transcription regulation and three-dimensional chromatin organization. In this protocol, we address the need for a quantitative means of investigating phase-separation mechanisms of Pol II transcription and CTCF functioning. We describe steps for protein purification, droplet formation, and automeasuring droplet properties. We then detail quantification during Pol II CTD and CTCF DBD clustering and outline their limitations. For complete details on the use and execution of this protocol, please refer to Wang et al. (2022) and Zhou et al. (2022)..