Why can't spike-in DNA be used in CUT&Tag assays?

Tagmentation takes place within the nuclei. To minimize non-specific background, any excess Tn5 enzyme is washed away prior to activation. Similarly, spike-in DNA introduced into the reaction would be eliminated alongside excess Tn5 if added before the initiation of tagmentation. Once tagmentation is complete, there is no benefit in adding spike-in DNA to the samples. This is because the spike-in DNA doesn't undergo tagmentation alongside antibody-targeted genomic DNA fragments. As a result, during PCR amplification of the CUT&Tag DNA library, the spike-in DNA would be lost due to the absence of adaptor sequences on its ends. Consequently, spike-in DNA is not compatible with the CUT&Tag assay.

If a spike-in strategy is desired, you can incorporate an equal amount of pre-isolated nuclei from another genome along with an antibody specifically targeting that genome in your testing reactions, for sample normalization purposes. Alternatively, you may consider choosing the CUT&RUN assay, which is compatible with spike-in DNA.

Last updated: May 1, 2024