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The Use of Flow Cytometry to Assess the State of Chromatin in T Cells
Published on: December 17, 2015
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Analysis of RNA Polymerase II Chromatin Binding by Flow Cytometry
Lilli T E Bay1, Trond Stokke1,2, Randi G Syljuåsen1
1Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, 0379 Oslo, Norway.
Bio-Protocol
|April 28, 2023
Summary
This study introduces a flow cytometry method to detect RNA polymerase II (RNAPII) and its phosphorylated forms in single human cells. This technique offers insights into transcription and DNA damage responses, overcoming limitations of bulk analysis methods.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- RNA polymerase II (RNAPII) is crucial for mRNA transcription and DNA damage responses in eukaryotic cells.
- Post-translational modifications, specifically serine 5 and serine 2 phosphorylation of RNAPII's C-terminal domain, mark distinct stages of transcription (promoter-proximal and elongating).
- Existing methods like ChIP-seq and chromatin fractionation often rely on large cell populations, potentially obscuring cell-cycle-dependent variations and cellular heterogeneity.
Purpose of the Study:
- To present a detailed flow cytometry protocol for detecting chromatin-bound RNAPII and its serine 5- and serine 2-phosphorylated forms in individual human cells across the cell cycle.
- To offer a single-cell analysis method that complements existing bulk techniques for studying RNAPII dynamics.
- To enable deeper understanding of transcription regulation and DNA damage responses at the single-cell level.
Main Methods:
- Development and validation of a flow cytometry protocol for intracellular staining.
- Detection of total RNAPII, serine 5-phosphorylated RNAPII, and serine 2-phosphorylated RNAPII.
- Analysis of RNAPII chromatin binding and phosphorylation states in individual human cells throughout the cell cycle.
Main Results:
- The protocol enables quantitative, single-cell analysis of RNAPII chromatin binding and its specific phosphorylation states.
- The method allows for the study of RNAPII dynamics in response to stimuli, such as ultraviolet DNA damage.
- The technique can reveal new insights into the transcription cycle and heterogeneity within cell populations.
Conclusions:
- The developed flow cytometry method provides a powerful tool for studying RNAPII function in individual cells.
- This approach overcomes the limitations of bulk methods by preserving information on cellular heterogeneity and cell cycle status.
- The protocol is envisioned as a valuable complementary technique to sequencing-based methods for investigating RNAPII-mediated transcription.
Keywords:
BarcodingCell cycleExtractionFlow cytometryMultiplexingRNA polymerase IIRNAPIITranscriptionMore Related Videos
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