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Updated: Aug 4, 2025
Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events
Published on: May 13, 2019
Droplet formation assay for investigating phase-separation mechanisms of RNA Pol II transcription and CTCF
Hui Wang1, Rong Zhou2, Xiong Ji2
1Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education, School of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China; Department of Pathogenic Biology, Chengdu Medical College, Chengdu 610500, China.
Abstract:
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)1 and Zhou et al. (2022).2.
