How CUT&RUN Profiles Chromatin | Cell Signaling Technology

The narrator explains that protein-DNA interactions drive biological processes like gene expression and cell differentiation. Understanding when and where these interactions occur is essential for studying epigenetics in normal development and disease.

Traditional ChIP-seq requires three days of work including chromatin crosslinking, fragmentation, immunoprecipitation, and DNA purification. CUT&RUN offers a faster alternative method with distinct advantages over the conventional approach.

Harvest cells and bind them to magnetic beads to simplify handling and minimize sample loss. Permeabilize the cells with digitonin—no crosslinking fixative is needed, allowing chromatin to remain in its native state.

Add antibodies targeting your protein of interest (transcription factors, cofactors, or histone modifications) to the permeabilized cells. Then add the enzyme Protein A-Protein G-Micrococcal Nuclease complex, which binds to the antibody through protein A or G interactions.

Add calcium to activate the micrococcal nuclease enzyme, which cuts DNA at sites where proteins interact with chromatin. This design provides precise targeting and effective control of nuclease activity.

The DNA-containing complexes diffuse freely into the supernatant, while remaining chromatin is retained in the cell nucleus. This separation greatly simplifies subsequent DNA purification steps.

Add spike-in control DNA to ensure experimental reproducibility. CUT&RUN generates lower background with less starting material compared to ChIP-seq and requires fewer sequencing reads, completing analysis in just one to two days.

Choose from Cell Signaling Technology's flexible CUT&RUN options: complete detection kits with all buffers and controls, or individual reagents and enzymes. Add validated primary antibodies specific to your target protein to complete the experiment.