ChIP-Seq Spike-in

a novel ChIP-Seq data normalization strategy


A persistent problem with chromatin immunoprecipitation is that traditional ChIP-Seq protocols are not always able to detect global changes in histone modifications caused by treatment with small molecule epigenetic inhibitors.

Active Motif's ChIP-Seq experts have developed and validated a solution for this problem and this novel spike-in and normalization strategy is now included in our end-to-end ChIP-Seq Service.

Review our recent PLoS One publication about this technique

  • An alternative approach to ChIP-Seq normalization enables detection of genome- wide changes in histone H3 lysine 27 trimethylation upon EZH2 inhibition.
    Brian Egan et al.

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How does it work?

ChIP-Seq reactions:

  • A standard ChIP-Seq reaction is set up using experimental chromatin (e.g. human) and an antibody of interest (e.g. H3K27me3 antibody).
  • In addition, Drosophila melanogaster chromatin is added, or “spiked-in”, to each reaction as a minor fraction of total chromatin.
  • An antibody that recognizes the Drosophila-specific histone variant, H2Av, is also added to the reaction, which provides a mechanism to reliably pull down a small fraction of Drosophila chromatin.
  • Following ChIP, immunoprecipitated DNA sequences are analyzed by Next-Generation Sequencing (NGS).

Data Analysis & Normalization

Once Next Generation sequencing is completed ...

  • Align sequence tags to the experimental reference genome (e.g. human) and the Drosophila genome.
  • Equalize differences in Drosophila tag counts across samples.
  • Count Human tags and then normalize using the same ratio used to equalize Drosophila tag counts.


Biases that are introduced during Next-Generation library amplification and sequencing also occur with the Drosophila spike-in chromatin. However, normalization using our Spike-in strategy eliminates these biases, enabling ChIP-Seq analysis to reveal any significant biological changes present in your samples.

ChIP-Seq Spike-in Normalization Strategy reveals changes in H3K27me3 levels following treatment with EZH2 inhibitor compound.
Figure 1: ChIP-Seq Spike-in Normalization Strategy reveals changes in H3K27me3 levels following treatment with EZH2 inhibitor compound.

Cells treated with small molecule inhibitors of the EZH2 methyltransferase have dramatic reductions in global H3K27me3 levels. However, H3K27me3 ChIP-Seq using standard ChIP-Seq protocols do not detect these differences (indicated by the - label above). Incorporation of Active Motif’s ChIP-Seq Spike-in strategy reveals the expected decrease in H3K27me3 ChIP-Seq signal (indicated by the + label above).