CUTANATM E. coli spike-in DNA (EpiCypher 18-1401) should be added in a consistent amount across all CUT&RUN reactions as a built-in normalization control. When used correctly, this small but powerful addition helps you get the most reliable and reproducible data from your CUT&RUN experiments.
What is the E. coli spike-in?
The E. coli spike-in consists of a low amount of Escherichia coli genomic DNA added directly to the digestion reaction. Because this DNA is not derived from your experimental sample, it provides an external reference point for downstream data normalization. When used consistently across experiments, E. coli spike-in reads serve as an internal scaling factor, enabling you to compare signal intensity across samples, time points, or experimental conditions.
Why it matters
In any CUT&RUN experiment, variation can arise from a variety of sources. The E. coli spike-in can help address many of these, including:
Sample handling or buffer prep downstream of fragment release
DNA recovery efficiency
Technical noise during library prep
Sequencing depth
Without a normalization reference, it’s difficult to know whether differences in read counts between samples reflect real signal or just technical variation. The E. coli spike-in solves this by providing a common yardstick: because the spike-in is introduced at known, consistent levels, any deviation in its read counts across samples can be interpreted as due to technical artifact, allowing you to adjust your conclusions accordingly. For example, if the proportion of reads aligning to E. coli in your reaction is above the recommended window (1-5%), this often indicates that your library is low complexity and/or many of your target reads were not retained. These factors are also associated with higher duplication rates. To learn more about how to calculate the E. coli normalization factor, check out this article.
When should you use spike-in normalization?
Use spike-in normalization if you need to:
Quantitatively compare signal between replicates
Evaluate major changes in global abundance across conditions
Confirm uniformity of library prep and sequencing
For qualitative comparisons (e.g. peak locations), spike-in normalization is less critical—but still beneficial to separate peaks from background noise (using IgG as a negative control). However, we recommend including E. coli in every reaction because it is a quick, easy way to add a powerful control to your experiment. Note: if you are concerned about E. coli consuming your sequencing reads, please refer to this article to ensure that you are optimizing your E. coli volume for the number of cells you are using.