Could you explain the techniques for normalization with yeast spike-in DNA and why it is performed for CUT&RUN assays?

A CUT&RUN assay allows for signal normalization across samples or experiments by either adding Drosophila spike-in nuclei with a Drosophila-specific H2Av antibody at the beginning of the experiment, or by adding yeast spike-in DNA in the stop buffer. Because the same amount of spike-in material is added to each sample, the signal from the spike-in genome should be consistent across all samples and experiments. Any variation in the spike-in signal therefore reflects technical noise, which can be corrected through normalization.

For example, if a sample is accidentally lost during processing or if fewer cells are used, a weaker signal may be observed. In such cases, the reduced signal does not represent a true decrease in protein–DNA binding. Spike-in–based normalization helps ensure that the measured signal more accurately reflects the real binding enrichment of the target protein across the genome.

Using yeast spike-in DNA allows normalization of variations introduced during DNA purification, library preparation, and next-generation sequencing, and it is a relatively low-cost option. Using Drosophila spike-in nuclei and antibodies enables normalization of variations introduced throughout the entire experimental workflow.

Both Drosophila and yeast spike-incontrols are optional. If you are not performing quantitative comparisons between datasets, spike-in materials are not strictly necessary.

Last updated: April 23, 2026