Single-Cell Factor Localization on Chromatin using Ultra-Low Input Cleavage Under Targets and Release using Nuclease

Area of Science:

• Molecular Biology
• Genomics
• Epigenetics

Background:

• Protein-DNA interactions are vital for gene regulation.
• Chromatin Immunoprecipitation (ChIP) is a traditional method, but antibody tethering techniques offer higher sensitivity.
• Cleavage Under Targets & Release Under Nuclease (CUT&RUN) is an antibody tethering method for mapping protein-DNA interactions.

Purpose of the Study:

• To present a detailed protocol for single-cell transcription factor profiling using an optimized CUT&RUN method.
• To enable the analysis of protein localization on chromatin in low cell populations and single cells.

Main Methods:

• Utilized recombinant Protein A tethered to micrococcal nuclease (pA-MNase) for site-specific DNA cleavage.
• Adapted CUT&RUN for ultra-low input conditions (uliCUT&RUN).
• Developed a manual 96-well format protocol for single-cell analysis.

Main Results:

• The uliCUT&RUN protocol allows for sensitive genome-wide profiling of transcription factors.
• The method is effective for both euchromatic and heterochromatic regions.
• Successfully optimized for single-cell resolution.

Conclusions:

• Ultra-low input CUT&RUN (uliCUT&RUN) is a powerful and sensitive technique for single-cell transcription factor profiling.
• This method enhances our ability to study protein localization and function in complex biological systems.
• The detailed protocol facilitates broader application of single-cell epigenomic analysis.