Could you explain the techniques for normalization of DNA and why it is performed for CUT&RUN assays?

A CUT&RUN assay allows signal normalization among samples or among experiments by adding spike-in DNA in the stop buffer. Since you add the same amount of spike-in DNA in the assay, the signal from spike-in DNA should be the same among all samples or experiments. Any variation of the spike-in DNA signals is due to technical errors that you can exclude by using the normalization strategy. For example, if you accidentally spill one of your samples during the experiment, or if you see lower signal with fewer starting cells, you know that the weaker signal does not represent the real binding strength of the protein on DNA in these scenarios. The sample normalization strategy can help to represent the real binding enrichment of a target protein on genome. Variation of signal strength introduced during DNA purification, library prep, and NGS can be normalized. That said, the spike-in DNA is totally optional. If you are not quantitively comparing a dataset to any other datasets, you do not need to use the spike-in DNA.
 

Last updated: September 12, 2024