Single Cell Cut&run

The antibody provides the targeting step: it binds the selected histone modification or DNA-binding protein, while the tethered micrococcal nuclease remains positioned near that chromatin target. Adding calcium activates the nuclease, allowing it to cleave nearby DNA. Because released fragments reflect this localized cleavage, sequencing can connect the selected chromatin feature with individual-cell regulatory states.

Analyzing cells individually preserves regulatory differences that would otherwise be averaged across a mixed sample. Cell-resolved profiles can therefore distinguish distinct chromatin states among cells sharing the same sample, helping identify cellular heterogeneity and relate epigenetic patterns to cell identity. This resolution is especially relevant when rare populations or developmental subgroups are present.

The assay can be directed toward specific histone modifications or DNA-binding proteins. That choice determines which chromatin-associated feature is mapped in each cell, allowing researchers to examine different aspects of epigenetic state and transcriptional regulation. Selecting the target according to the biological question helps connect the resulting DNA fragments with a particular regulatory process.

The relatively low input requirement makes the approach useful when only limited biological material is available. It can support analysis of rare cell populations that may be obscured in larger mixed samples, while still preserving cell-to-cell differences. In biology studies, this capability helps investigate heterogeneous tissues, developmental samples, and disease states without relying only on abundant populations.

The workflow starts by permeabilizing cells so the antibody can access chromatin. The antibody is then applied to the chosen histone modification or DNA-binding protein, and the tethered micrococcal nuclease is activated with calcium. Nearby DNA is cleaved and released, after which the fragments are indexed by cell and prepared for sequencing to generate cell-resolved profiles.

Cell indexing preserves the origin of each released DNA fragment during sequencing. Researchers can therefore assign chromatin-associated fragments back to individual cells rather than interpreting only a combined sample signal. This assignment enables comparisons of regulatory states across cells and supports links between chromatin features, cellular identity, and transcriptional regulation.

Single-cell CUT&RUN is useful for studying cellular heterogeneity, development, disease states, and rare cell populations. Its ability to connect cell identity with epigenetic state provides context that bulk measurements may not resolve. The resulting chromatin profiles can also complement single-cell transcriptomic analyses, allowing regulatory features to be considered alongside cell-specific gene-expression information.