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A fluorescence-based protocol to quantitatively titrate CUT&RUN buffer components.

Andrew Katznelson¹, Kenneth Zaret¹

¹Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

|February 8, 2024

Summary

This study introduces a fluorescence-based method to optimize Cleavage under targets and release using nuclease (CUT&RUN) buffer conditions. The protocol ensures efficient cell permeabilization and epitope retention for accurate genomic analysis.

Keywords:

Cell Biology Cell-based Assays ChIP-seq Chromatin immunoprecipitation (ChIP)Genetics Molecular Biology

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Area of Science:

Molecular Biology
Genomics
Biochemistry

Background:

Cleavage under targets and release using nuclease (CUT&RUN) is a key technique for mapping protein-DNA interactions and histone modifications in chromatin.
Optimizing buffer conditions is crucial for efficient cell permeabilization and maintaining epitope integrity during CUT&RUN assays.

Purpose of the Study:

To develop and present a fluorescence-based protocol for quantitative titration of CUT&RUN buffer components.
To optimize cell permeabilization and target epitope retention on chromatin for improved CUT&RUN assay performance.

Main Methods:

Cells were captured on concanavalin A beads.
A fluorescence-based assay was employed to titrate digitonin and NaCl concentrations in CUT&RUN buffers.
Fluorescence imaging was used to determine optimal buffer conditions.

Main Results:

The developed protocol allows for quantitative assessment of CUT&RUN buffer components.
Optimal concentrations of digitonin and NaCl were identified to enhance cell permeabilization and epitope retention.
The fluorescence-based titration provides a robust method for assay optimization.

Conclusions:

This fluorescence-based titration protocol offers an efficient and quantitative approach to optimize CUT&RUN assays.
The optimized conditions enhance the reliability and accuracy of identifying genomic binding sites for proteins and histone modifications.