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.
Learn about our new ChIP-Seq Spike-in strategy
How does it work?
- 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.
Figure 1: ChIP-Seq Spike-in Normalization Strategy reveals changes in H3K27me3 levels following treatment with EZH2 inhibitor compound.