Is CUT&Tag assay compatible with spike-in sample normalization?

Yes. We recommend the Drosophila spike-in normalization approach, which enables reliable and consistent normalization across samples, corrects for variations in starting cell number, technical variation among samples during processing, and non-specific enrichment coming from isotype control antibodies. Detailed instructions for implementing spike-in controls are provided in our Drosophila Spike-In Control Kit for CUT&Tag (Rabbit) #29811 or Drosophila Spike-In Control Kit for CUT&Tag (Mouse) #19629.

With the Drosophila spike-in strategy, Drosophila nuclei are added concurrently with the test cells at the start of the CUT&Tag workflow, ensuring they experience all the same steps and conditions as the test cells. The H2Av Rabbit or Mouse Monoclonal Antibody, which specifically recognizes Drosophila chromatin, is included along with the test antibody. A normalization factor is then generated from the Drosophila enrichment signal and applied to the test genome to normalize signal across samples. This approach normalizes all steps throughout the entire protocol.

Some protocols rely on contaminating E. coli DNA present in pAG-Tn5 for normalization, but this can lead to inconsistencies when switching enzyme lots, as the amount of contaminating DNA varies. The Drosophila spike-in strategies provide better lot-to-lot consistency and more reliable normalization.

Last updated: April 23, 2026