The Spike-in Normalization Strategy
Description
The Spike-in Antibody (Catalog No. 61686) is intended for use with Active Motif’s Spike-in Chromatin (Catalog No. 53083) to normalize
chromatin immunoprecipitation (ChIP) experiments. ChIP is a multi-step process in which variations caused by sample loss during immunoprecipitation and library preparation, uneven sequencing read depth or user differences can lead to results that are difficult to interpret. To
overcome this challenge, Active Motif has developed a spike-in strategy to normalize out technical variation and sample processing bias.
Additionally, the normalization strategy can be used to monitor the effects of experimental conditions, such as inhibitory compounds ormutants.
A standard ChIP reaction is set up using experimental chromatin (e.g. human) and an antibody of interest. In addition, Drosophila melanogaster chromatin (Spike-in 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 also spiked-in to the reaction (Spike-in Antibody). This Spike-in antibody
provides a mechanism to reliably pull down a small fraction of Drosophila chromatin that is consistent across all samples. Since variation
introduced during the ChIP procedure will also occur with the spike-in chromatin, a normalization factor can be created based on theDrosophila signal and applied to the sample genome. This ChIP Spike-in strategy enables normalization of data across ChIP antibodies andexperiments without bias.
Products• Spike-in Antibody (Catalog No. 61686)• Spike-in Chromatin (Catalog No. 53083)• 96 rxns Drosophila Positive Control Primer Set Pbgs (Catalog No. 71037)• 96 rxns Drosophila Negative Control Primer Set 1 (Catalog No. 71028)• ChIP-IT High Sensitivity Kit (Catalog No. 53040)• ChIP-IT qPCR Analysis Kit (Catalog No. 53029)

Experimental
Chromatin

Spike-in
Chromatin
Antibody ofinterest

Spike-in
Antibody

Chromatin Immunoprecipitation

Sequencing

Map to experimental
genomeNormalize sample
tag counts by same ratio

Map to Drosophila
genomeNormalize Drosophila
tag counts across samples

ChIP-Seq Normalization Workflow

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Guidelines
Active Motif’s normalization strategy may be applied to any mammalian ChIP reaction due to the low cross-reactivity of the Spike-in
Antibody with mammalian samples. Researchers may choose to optimize the amount of sample chromatin and antibody used per ChIP
reaction. However, we recommend adjusting the amount of Spike-in Chromatin to maintain the same chromatin ratio (sample : spike-in) as
recommended on the lot-specific Spike-in Chromatin data sheet that accompanies the purchase of the product.
ChIP Spike-in Reaction Guidelines
Samplechromatin

Spike-inchromatin

Antibodyof interest

Spike-inAntibody

Robust antibodies against abundant histone modifications

25 µg

Refer to lot-specificdata sheet

4 µg

2 µg

Antibodies against transcription factors, histone modifiers orlow abundance histone modifications

25 µg

Refer to lot-specificdata sheet

4 µg

2 µg

Simply add the Spike-in Chromatin and Spike-in Antibody to your standard ChIP reaction containing sample chromatin and the antibody ofinterest. Use the recommended ratio based on the antibody target. Perform ChIP as normal. Follow the guidelines to normalize the data for
qPCR or ChIP-Seq analysis.

The Spike-in Antibody shows minimal
cross reactivity with mammaliansamples. When the Spike-in Antibody
was tested in ChIP-Seq with humanchromatin, there is little to no signal
detected. This demonstrates thespecificity of the Spike-in Antibody
for the Spike-in Chromatin ensuring
low background during ChIP.

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

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The following guidelines are provided for qPCR normalization:
1. Perform ChIP combining the Spike-in Chromatin, Spike-in Antibody, sample chromatin and antibody of interest into the same tube
for immunoprecipitation. Use the guidelines provided for chromatin and antibody quantities based on the antibody target.
2. Following purification of the ChIP DNA, perform qPCR using primers designed to amplify the experimental region of interest. The
same ChIP DNA is also used in qPCR with Drosophila Positive control Primer Set for the Pbgs gene (Catalog No. 71037) to determine
the amount of Spike-in present in each sample.
3. Use data from one of the Drosophila positive control IP reactions to determine a normalization factor that equalizes the signal
across samples. Always use a positive primer set as the selected data point for normalization as the negative primers should havevery low signal.		 (Selected Sample / Selected Sample) = Normalization factor of 1		 (Selected Sample / Sample 2) = Normalization Factor X for IP 2		 (Selected Sample / Sample 3) = Normalization Factor Y for IP 3
4. Normalize the experimental samples by multiplying each qPCR signal by it’s corresponding normalization factor. These adjustedvalues represent the normalized data.
Below is an example of qPCR normalization:
Spike-In AntibodyBinding events detected per 1000 cell values

Example Data for qPCR Normalization
Normal IP

50% Beads

50% Loss

Drosophila Negative Control Primer Set

232018

Drosophila Positive Control Primer Set Pbgs

180125110

Antibody of Interest
Binding events detected per 1000 cell values

Example Data for qPCR Normalization
Normal IP

50% Beads

50% Loss

Human Negative Control Primer Set

0.5

0.4

0.4

Human Positive Control Primer Set

352422

To normalize the data, select a positive control sample from the Spike-in Antibody and use this value to create normalization
factors for the rest of the data. In this example select the Normal IP value of 180. Divide the selected value by each spike-in
data point to create the normalization factor for each IP reaction.
Normalization Factor
Drosophila Positive Control Primer Set Pbgs

Normal IP

50% Beads

50% Loss

180 / 180= 1.0

180 /125= 1.44180 / 110
= 1.64

Adjust the antibody of interest data by multiplying each IP reaction by it’s corresponding normalization factor.
Antibody of Interest
Binding events detected per 1000 cell values

Normalized Data
Normal IP

50% Beads

50% Loss

Human Negative Control Primer Set0.5x 1.0 =0.5

0.4 x 1.44 =0.6

0.4 x 1.64 =0.7

Human Positive Control Primer Set

35 x 1.0 =35

24 x 1.44 =34.6

22 x 1.64 =36.1

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The following guidelines are provided for ChIP-Seq normalization:
1. Perform ChIP combining the Spike-in Chromatin, Spike-in Antibody, test chromatin and test antibody into the same tube for immunoprecipitation. We suggest using the guidelines provided for chromatin and antibody quantities based on the antibody target.
2. Follow ChIP with Next-Generation Sequencing.
3. Map ChIP-seq data to the test reference genome (e.g. human, mouse or other).
4. Map ChIP-seq data to the Drosophila reference genome.
5. Count uniquely aligning Drosophila sequence tags and identify the sample containing the least number of tags.
6. Compare Drosophila tag counts from other samples to the sample containing the least tags and generate a normalization factor foreach comparison.		 (Sample 1 with lowest tag count / Sample 2) = Normalization factor
7. Downsample the tag counts of data sets proportional to the normalization factor determined.
Below is an example of ChIP-seq normalization:
Drosophila alignments

Sample 1

Sample 2

Sample 3

Total number of reads

36,024,410

41,210,918

50,337,645

Unique alignments

4,695,354

5,182,606

3,164,431

Final number of tags without duplicate reads

4,039,904

4,136,364

2,281,096

Sample genome alignments

Sample 1

Sample 2

Sample 3

Total number of reads

36,024,410

41,210,918

50,337,645

Unique alignments

21,533,256

21,261,810

33,382,716

Final number of tags without duplicate reads

19,221,841

17,600,478

24,839,738

To normalize the data, select the sample with lowest number of Drosophila final tags and use this value to create normalization
factors for the rest of the data. In this example select 2,281,096. Divide the selected value by the Drosophila tag counts fromthe other samples to create the normalization factor for each IP reaction.
Normalization Factor
Drosophila final tags without duplicate reads

Sample 1Sample 2

Sample 3(2,281,096 / 4,039,904)= 0.5646(2,281,096 / 4,136,364)= 0.5515(2,281,096 / 2,281,096)= 1.0

Adjust the sample genome final tag counts by multiplying each sample by it’s corresponding normalization factor.
Normalized Data
Sample final tags without duplicate reads

Sample 1

Sample 2

Sample 3(19,221,841 x 0.5646) =10,853,442(17,600,478 x 0.5515) =9,706,201(24,839,738 x 1.0) =24,839,738

Downsample the tags from each data set to achieve the normalized tag counts.

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Technical Services
If you need assistance at any time, please call Active Motif Technical Service at one of the numbers listed below.
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FAQs
Q1. Spike-in Chromatinは固定化されていますか？
 はい。ホルムアルデヒドにより固定化済みです。
Q2. Spike-in Chromatinは断片化されていますか？
 はい。ソニケーターを用いて断片化済みです。
Q3. Spike-inの断片化サイズはどのくらいですか？
 200-1200 bpになります（多少のバッチ間による差はあります）。
Q4. Spike-in Antibodyは何由来の抗体ですか？
 ウサギ由来のポリクローナル抗体です。
（改訂日 2020.9）

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