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ChIP Normalization

reduce unwanted variation to reveal latent biological effects

ChIP Normalization reveals changes in H3K27me3 levels following treatment with EZH2 inhibitor.
ChIP Normalization reveals reduction in H3K27me3 caused by an EZH2 inhibitor.

 

Active Motif's ChIP Spike-in Normalization Strategy can be used to normalize ChIP qPCR and ChIP-Seq data to reduce the effects of technical variation and sample processing bias. ChIP is a multi-step process in which variations caused by sample loss, uneven sequencing read depth or user differences can lead to results that are difficult to interpret. Active Motif's Normalization Strategy can be applied across samples and antibodies to eliminate this bias and reveal any latent biological effects induced by causal agents such as inhibitory compounds or mutants.

ChIP normalization can be implemented simply by integrating our Spike-in reagents into your standard ChIP protocol. A standard ChIP reaction is set up using experimental chromatin and an antibody of interest. Spike-in Chromatin and Spike-in Antibody are also added, or 'spiked in', to the ChIP reaction as a minor fraction of the IP reaction. Any variation introduced during the ChIP reaction will also occur with the Spike-in chromatin. Since the Spike-in chromatin is consistent across all samples, a normalization factor to be created based on the Spike-in signal and applied to the sample genome.

ChIP Spike-in Normalization Advantages

  • Reduce the effects of technical variation
  • Detect latent or subtle biological differences that are not observed with standard ChIP analysis
  • Can be applied across different antibodies and samples without bias
  • Spike-in Chromatin and Spike-in Antibody can be used with any ChIP protocol
  • Strategy works with both qPCR and ChIP-Seq analysis

To learn more about the ChIP Normalization Strategy, click on the Method, Data, or Contents tabs below. To view a protocol or other related documents, click on the Documents tab below.

 
Name Format Cat No. Price  
Spike-in Antibody 50 µg 61686 $175 Buy Now
Spike-in Chromatin 15 rxns 53083 $155 Buy Now
Drosophila Positive Control Primer Set Pbgs 96 rxns 71037 $90 Buy Now
Drosophila Negative Control Primer Set 1 96 rxns 71028 $90 Buy Now

ChIP Spike-in Normalization Advantages

  • Enables normalization of ChIP results to reduce the effects of technical variation
  • Detect subtle biological differences that are not observed with standard ChIP analysis
  • Apply normalization strategy across different antibodies and samples without bias
  • Spike-in Chromatin and Spike-in Antibody can be used with any ChIP protocol
  • Normalization strategy works with both qPCR and ChIP-Seq analysis

How does the ChIP Spike-in Normalization Strategy work?

A standard ChIP reaction is set up using experimental chromatin and an antibody of interest. Spike-in chromatin and a Spike-in Antibody are also added to the reaction. The Spike-in Antibody recognizes a histone variant that is specific to the species of the Spike-in chromatin (Drosophila). This enables specific detection of the spike-in chromatin without any significant increase in background signal. Since variation introduced during the ChIP procedure will also occur with the spike-in chromatin, a normalization factor can be created from the spike-in chromatin and applied to the experimental chromatin to normalize out technical variation and sample bias, or to monitor biological effects.

  

  Sample Chromatin Spike-in Chromatin Antibody of Interest Spike-in Antibody
ChIP Spike-in Reaction Guidelines
Robust antibodies against abundant histone modifications  25 µg  Refer to lot-specific data sheet  4 µg  2 µg
Antibodies against transcription factors, histone modifiers or low abundance histone modifications  25 µg  Refer to lot-specific data sheet  4 µg  2 µg

 

Flow Chart of the ChIP Normalization Strategy for ChIP-Seq from Active Motif
Figure 1: ChIP-Seq Normalization Workflow.

A standard ChIP reaction is set up using experimental chromatin (e.g. human) and an antibody of interest. In addition, Drosophila melanogaster chromatin is added, or "spiked-in" to each reaction as a minor fraction of the total chromatin. An antibody that recognizes the Drosophila-specific histone variant, H2Av, is added to the reaction. The Spike-in antibody provides a mechnaism to reliable pull down a small fraction of the Drosophila chromatin that is consistent across all samples. Following ChIP sequencing, the the data is mapped to both the Drosophila genome and the experimental genome. A normalization factor is created for each sample based on the Drosophila tag counts. The experimental tag counts are normalized by the same factor.

Reduce the Effects of Technical Variation

The ChIP Normalization Strategy is ideal to correct for differences that results from sample loss, amplification bias, uneven sequencing read depth or hand-to-hand differences between users. By utilizing the differences observed between samples with the Spike-in chromatin, a normalization factor is created and applied to the experimental samples to normalize out the effects of technical variation.

ChIP qPCR analysis using the Spike-in chromatin
ChIP qPCR analysis using human chromatin
Normalized ChIP qPCR data using the Spike-in Normalization Strategy
Figure 1: ChIP Normalization of technical variation by ChIP qPCR.

A ChIP qPCR reaction was set up with 750 ng of Spike-in chromatin added to 30 µg of human chromatin. Both Spike-in Antibody (2 µg) and a Histone H3K27me3 antibody (4 µg) were also added to the ChIP reaction. ChIP was performed according to Active Motif's ChIP-IT High Sensitivity protocol (Catalog No. 53040), however, one sample was left as normal, one sample was given only 50% of the Protein G beads and the third sample had 50% of the IP volume removed. Enriched DNA was analyzed by qPCR using both Drosophila-specific primers and Human-specific primers for each sample. The results show the effects of these sample differences on the enrichment. The Drosophila data was used to create a normalization factor for each sample. This normalization factor was then applied to each Human data point. The normalized data reveals comparable ChIP qPCR results following the removal of technical variation effects, validating the Spike-in strategy for normalization.


Identify Biological Differences Not Observed by Standard ChIP Analysis

By adding Spike-in Chromatin and Spike-in Antibody to standard ChIP reactions, experimental data can be normalized for sample variation. This normalization makes it easier to monitor the effects of experimental conditions, such as inhibitory compounds or mutants to reveal biological differences.

PCR analysis showing specific enrichment from low abundance target proteins
Figure 2: Normalization of biological differences in ChIP-Seq.

ChIP-Seq was performed on untreated cells and cells treated with a small molecule inhibitor of EZH2 methyltransferase. Using standard ChIP-Seq analysis (–) the differences in signal are not detected. Incorporation of the Spike-in Normalization Strategy (+) reveals the expected decrease in H3K27me3 ChIP-Seq signal confirming the value of the normalization strategy for detecting biological changes.


Specificity of Detection

The Spike-in chromatin consists of Drosophila melanogaster chromatin prepared from Schneider's Drosophila Line 2 (S2) cells. The Spike-in antibody recognizes a Drosophila-specific Histone variant, H2Av. Because of the specificity of the Spike-in antibody for the Spike-in chromatin modification, there is no cross-reactivity with mammalian samples leading to reduced background signal.

Specificity of the Spike-in Antibody
Figure 3: Specificy of the Spike-in Antibody.

The Spike-in antibody shows minimal cross reactivity with mammalian samples. When the Spike-in antibody was tested in ChIP-Seq with human chromatin, there is little to no signal detected. This demonstrates the specificity of the spike-in normalization strategy.

Contents & Storage

Please note that the ChIP Normalization reagents are available separately. Both the Spike-in Chromatin and Spike-in Antibody are required to apply the normalization strategy. Drosophila-specific qPCR primer sets are available for ChIP qPCR analysis.

Spike-in Antibody

  • 50 µg Spike-in Antibody supplied at a concentration of 1 µg/µl in PBS containing 0.035% sodium azide and 30% glycerol

Spike-in Chromatin

  • Spike-in Chromatin prepared from Schneider's Drosophila Line 2 (S2) cells is provided for 15 rxns of robust histone modification antibody targets. Spike-in chromatin is provided at a concentration of 10 ng/µl.

qPCR Primer Sets

  • 400 µl Drosophila Positive Control Primer Set Pbgs is supplied at a concentration of 2.5 µM
  • 400 µl Drosophila Negative Control Primer Set 1 is supplied at a concentration of 2.5 µM