Spike-In Normalization: Complete Guide
Enabling Epigenetics Research

Overview, Methods, and Resources

Spike-In Normalization: Complete Guide

Overview, Methods, and Resources

Introduction

Identifying differences between epigenetic assay data sets canbe challenging when global modification changes occur, such as
when studying the effects of chromatin modifying enzyme inhibitors.
Additionally, inaccurate quantification of starting material or technicalvariation during processing results in variation across sample data.
Currently available bioinformatic-based normalization methods are notapplicable for normalizing across data sets in these instances, and the only
reliable way to overcome bias and variation is to add a known standard(Spike-In) into all samples. Specific Spike-In reagents for ATAC-Seq, ChIPSeq, CUT&RUN, and CUT&Tag are invaluable tools that overcome data
variation challenges, allowing researchers to see, compare, and reporttrue biological differences between samples in each of these assays.
With this eBook, you will find a complete resource for normalizationof assay data sets – from the background and publication of
Spike-In normalization to sample data and comparisons, tools andreagents available, and educational resources for further study.

Introduction | i

Spike-In Normalization: Complete Guide

Overview, Methods, and Resources

Table of Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i
Overview of Spike-In Normalization . . . . . . . . . . . . . . . . . . . 1
ChIP-Seq Spike-In . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  2
ChIP Spike-In Normalization Advantages
How Can Spike-In Normalization Improve ChIP-Seq Analysis?

ATAC-Seq Spike-In . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  6
ATAC-Seq Spike-In Control Advantages

CUT&RUN Spike-In . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  7
Overview
CUT&RUN Spike-In Control Advantages

CUT&Tag-IT® Spike-In . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
How Does CUT&Tag-IT Spike-In Control Work?
CUT&Tag-IT® Spike-In Control Advantages

CUT&Tag-IT® R-loop Spike-In . . . . . . . . . . . . . . . . . . . . . . .  15
CUT&Tag-IT® Spike-In Control, R-loop Advantages

Resources & Ordering Information . . . . . . . . . . . . . . . . . .  17
Application Resource Centers
Ordering Information
Webinar

Table of Contents |ii

Spike-In Normalization: Complete Guide

Overview, Methods, and Resources

Overview of Spike-In Normalization
It began with the original publication of this method by Brian Egan et al.,An Alternative Approach to ChIP-Seq Normalization Enables Detectionof Genome-Wide Changes in Histone H3 Lysine 27 Trimethylation
upon EZH2 Inhibition (PLoS One. 2016).1 This important work focusedon ChIP-Seq, which is widely used for the detection of transcription
factor binding patterns and histone post-translational modification(PTM) occupancy profiles across the entire genome. ChIP-Seq datafrom many different cell types and contexts are used to generate
genome-wide chromatin modification maps to explore the relationshipbetween transcriptomic and epigenomic landscapes, and cell typecomparisons often reveal substantial lineage-related differencesin the profiles of specific histone PTMs across genomes. However,when there is manipulation of biological context, e.g. comparisons ofknockdown or knockout of individual histone modifying enzymes ortheir inhibition with small molecules, these conditions can contributevery subtle alterations to the PTM landscape rather than resulting in
a completely different, more easily detectable patterns. In an effort toaddress this, more complex statistical methods, software programs andcomputational models have been developed in an attempt to adequately
compare ChIP-Seq data sets and reliably reveal the differences.
A previous technique, ChIP-Rx, based on the addition of a constantamount of reference cells from a different species, had been publishedfor genome-wide quantitative comparison of histone modifications
across different biological samples.2 The method depends on the

1.

Egan B, Yuan CC, Craske ML, Labhart P, Guler GD, Arnott D, Maile TM, Busby J, Henry C, et al. An Alternative
Approach to ChIP-Seq Normalization Enables Detection of Genome-Wide Changes in Histone H3 Lysine 27
Trimethylation upon EZH2 Inhibition. PLoS ONE 11(11): e0166438. PMID: 27875550

ability of the experimental ChIP antibody to recognize the histone
modification of interest in both the reference and experimental
cell species. Precipitated reference DNA is sequenced along with
precipitated experimental DNA, and thus reference sequence reads
provide a means to normalize across biological samples. Egan’s
team developed an alternative normalization approach based on the
addition of Drosophila melanogaster chromatin and a D. melanogasterspecific antibody into standard ChIP reactions. Specifically, the use ofan antibody that exclusively recognizes the D. melanogaster histone
variant H2Av enables precipitation of D. melanogaster chromatin as aminor fraction of the total ChIP DNA. The D. melanogaster ChIP-Seq
tags are used to normalize the human ChIP-Seq data from samples.
One particularly compelling example of the importance of this ChIP-Seq
normalization strategy is illustrated in their experiments investigating
the genome-wide localization of the repressive histone mark H3K27me3
in the presence of epigenetic inhibitors. H3K27me3 has one identified
methyltransferase (EZH2) responsible for catalyzing methyl group
addition. However, EZH2 inhibition did not appear to significantly affect
H3K27me3 levels in a ChIP-Seq experiment. Following normalization to
Spike-In, a substantial reduction in H3K27me3 signal was then observedin ChIP-Seq data from EZH2 inhibitor treated samples, demonstrating
how the use of Spike-In normalization can reveal changes in histone
modifications that would otherwise be masked by experimental artifacts.

2.

Orlando DA, Chen MW, Brown VE, Solanki S, Choi YJ, Olson ER et al. Quantitative ChIP-Seq Normalization
Reveals Global Modulation of the Epigenome. Cell Reports. 2014;9(3):1163–1170. PMID:25437568

Overview of Spike-In Normalization |1

Spike-In Normalization: Complete Guide

Overview, Methods, and Resources

ChIP-Seq Spike-In

How Does ChIP-Seq Spike-In Work?
ChIP normalization can be easily implemented simply byintegrating Spike-In reagents into your standard ChIP protocol
using experimental chromatin and an antibody of interest. Spike-In
Chromatin and Spike-In Antibody are also added, or "spiked in", tothe ChIP reaction as a minor fraction of the IP reaction. Any variation
introduced during the ChIP reaction will also occur with the SpikeIn Chromatin. As the amount of spiked-in chromatin is consistentacross all samples, a normalization factor can be created basedon the Spike-In signal and applied to the experimental signal.
Spike-In chromatin and a Spike-In Antibody can easily be integrated
into an existing ChIP-Seq workflow. Spike-In Chromatin and the
Spike-In Antibody are added to experimental chromatin and theexperimental antibody just prior to immunoprecipitation. The
Spike-In antibody recognizes a histone variant that is specificto the species of the Spike-In Chromatin (Drosophila), and theexperimental antibody specifically recognizes the experimental
chromatin. This enables specific detection of the Spike-In Chromatinwithout any significant increase in background signal. Following
sequencing, reads will be mapped to their specific species. Variation
introduced during the ChIP procedure will affect the Spike-In
Chromatin in the same manner as the experimental chromatin, so
a normalization factor can be created from the Spike-In Chromatin
and applied to the experimental chromatin to normalize out technicalvariation and sample bias, or to monitor biological effects.

ChIP Spike-In Normalization Advantages⊲ Reduce effects of technical variation to detect subtle
biological differences not seen with standard ChIP
⊲ Can be applied across different antibodies
and samples without bias
⊲ Spike-In Chromatin and Spike-In Antibody canbe used with any ChIP kit or protocol⊲ Strategy works with both qPCR and ChIP-Seq analysis

ChIP-Seq Spike-In Normalization publications

ChIP-Seq Spike-In |2

Spike-In Normalization: Complete Guide

Overview, Methods, and Resources

ChIP-Seq Spike-In Uses Drosophila Chromatin
Drosophila
Chromatin

Drosophila
H2Av antibody

Targetantibody

Humanchromatin

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 melanogasterchromatin is added, or "spiked-in" to each reaction as a minor fraction of thetotal chromatin. An antibody that recognizes the Drosophila-specific histone
variant, H2Av, is added to the reaction. The Spike-In Antibody provides amechanism to reliably pull down a small fraction of the Drosophila chromatinthat is consistent across all samples. Following ChIP sequencing, 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.
normalization factor is created for each sample based on the Drosophila tag
counts. The experimental tag counts are normalized by the same factor.

ChIPStandard library amplification

Sequencing

Map to Drosophila
genome

Map to humangenomeNormalize Drosophila
tag counts across samples

Normalize human sample
tag by same ratio as Drosophila

ChIP-Seq Spike-In |3

Spike-In Normalization: Complete Guide

Overview, Methods, and Resources

Specificity of Detection
The Spike-In Chromatin consists of Drosophila melanogasterchromatin 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.
Figure 2: Specificity 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.

ChIP-Seq Spike-In | 4

Spike-In Normalization: Complete Guide

Overview, Methods, and Resources

H3K27me3 ChIP-Seq

How Can Spike-In Normalization
Improve ChIP-Seq Analysis?
One particularly compelling example of the importance of ChIP-Seq
normalization is illustrated in experiments investigating the genomewide localization of the repressive histone mark H3K27me3 in thepresence of epigenetic inhibitors. H3K27me3 has one identified
methyltransferase (EZH2) responsible for catalyzing methyl group
addition. However, EZH2 inhibition did not appear to significantly affect
H3K27me3 levels in a ChIP-Seq experiment without normalization to
Spike-In, in which the significant loss of H3K27me3 becomes apparent.
Therefore, Spike-In can reveal changes in histone modifications thatwould otherwise be masked by experimental artifacts (Figure 3).

Figure 3: Standard ChIP-Seq Methods Do Not Reveal
EZH2 Inhibition Without Spike-In Normalization
Left Westerm blot showing reduced global H3K27me3 levels in KARPAS-422
cells treated with 1.5 µM CPI-360 for 4 and 8 days. Whole cell extracts wereresolved by SDS-PAGE and immuno-blotted with anti-H3K27me3. Anti-H3
immuno-blots show equal levels of total H3. Right Representation of
H3K27me3 ChIP-Seq data using IGV. No obvious differences are detected in
CPI-360 treated KARPAS-422 cells when compared to KARPAS DMSO controls.

Egan B., et al., An Alternative Approach to ChIP-Seq Normalization Enables Detection of Genome-Wide Changes in
Histone H3 Lysine 27 Trimethylation upon EZH2 Inhibition. PLoS One, 2016. 11(11):e0166438

ChIP-Seq Spike-In| 5

Spike-In Normalization: Complete Guide

Overview, Methods, and Resources

ATAC-Seq Spike-In

Spike-In normalizes out differences in starting cell numbers
Assay for Transposase-Accessible Chromatin with high-throughput
sequencing (ATAC-Seq) has emerged as a powerful method forinvestigating open or accessible chromatin across the genome. However,identifying differences between data sets can be challenging when global
modification changes occur, such as studying the effects of chromatin
modifying enzyme inhibitors. Additionally inaccurate quantificationof starting material or technical variation during processing resultsin variation across sample data. Active Motif’s strategy for ATAC-Seq
normalization is to Spike-In cryopreserved Drosophila cell nuclei into
samples prior to nuclei prep and tagmention. During tagmentation boththe test cells and the Drosophila nuclei are tagged at open chromatin
consistently across all samples. A normalization factor is then createdbased on the Drosophila signal and applied to the test genome.

ATAC-Seq Spike-In Control Advantages⊲ Identify differences between datasets⊲ Reveal changes in open chromatin that weremasked by cell number differences⊲ Overcome bias and variation with known standard

Figure 4: K562 Starting Cell Numbers for ATACSeq with and without Spike-In Normalization
This experiment used 10,000 Drosophila nuclei as a spike in for a mocktreatment. The mock treatment was a cell titration performed in duplicate
from 200,000 cryopreserved K562 cells down to 10,000 cryopreserved K562
cells. The actual cell numbers are 200,000, 100,000, 50,000, 20,000 and
10,000 cells. This figure is both the spike-in normalized and unnormalized
tracks for one of the duplicates in the titration.

ATAC-Seq Spike-In| 6

Spike-In Normalization: Complete Guide

Overview, Methods, and Resources

CUT&RUN Spike-In

Overview
In CUT&RUN, a protein of interest is tagged with an antibody and boundto the chromatin in intact cells. A micrococcal nuclease (MNase) is used
to cleave the DNA specifically at the binding sites of the protein ofinterest. The released fragments are purified, sequenced, and mapped
to the reference genome to determine the protein’s binding sites. Unlike
ChIP, CUT&RUN does not require crosslinking of the protein to the DNA,
which can introduce artifacts. CUT&RUN is a valuable tool for studying
chromatin-associated proteins because it is sensitive, specific, andrequires fewer cells than ChIP, making it ideal for identifying binding
patterns of chromatin-associated proteins such as transcription factors
or histone modifications genome-wide. Chromatin-associated proteinsplay critical roles in regulating various cellular processes such as gene
expression, DNA replication, DNA repair, and cell differentiation.
Active Motif’s strategy for CUT&RUN normalization is to Spike-In
cryopreserved Drosophila cell nuclei into samples prior to CUT&RUN.
Then, during the antibody incubation step, a Drosophila H2Av antibody is
added in addition to the antibody to the target of interest. This Drosophila
H2Av antibody provides a mechanism to reliably tag Drosophila histones
in a consistent way across all samples. A normalization factor is thencreated based on the Drosophila signal and applied to the test genome.
This CUT&RUN Spike-In strategy enables normalization of CUT&RUN
data independent of the experimental antibody and without bias.

Cells

CUT&RUN
Spike-In Nuclei

Target-Specific
Antibody

CUT&RUN
Spike-In Antibody

CUT&RUN

Sequencing

Map to experimental
genome

Map to Drosophila
genomeNormalize Drosophila
tag counts across samples

Normalize experimental
sample tag counts
by same ratio

CUT&RUN Spike-In | 7

Spike-In Normalization: Complete Guide

Overview, Methods, and Resources

CUT&RUN Spike-In Control Data
Active Motif’s CUT&RUN normalization strategy may be applied toany mammalian CUT&RUN assay reaction due to the lack of crossreactivity of the Spike-In antibody with mammalian samples. The
amount of cryopreserved Drosophila nuclei and antibody used
per CUT&RUN reaction may need to be optimized with the goal of
having Drosophila reads make up only 5-10% of the total sequencing
reads. However, when using robust antibodies against tightly
localized histone modifications, such as H3K4me3, we recommenda Spike-In:test sample ratio of 1:20. For antibodies against spreading
marks such as H3K27me3, we recommend a Spike-In:test sample
ratio of 1:10. For antibodies against transcription factors suchas YY1, we recommend a Spike-In:test sample ratio of 1:100.

CUT&RUN Spike-In Control Advantages⊲ Compare between CUT&RUN datasets
between experimental samples⊲ Reveal differences masked by cell number inputs⊲ Simply add Spike-In Nuclei to samples and perform
CUT&RUN with the CUT&RUN Spike-In Antibody
together with experimental target antibody

CUT&RUN Spike-In| 8

Spike-In Normalization: Complete Guide

Overview, Methods, and Resources

Figure 5. K562 Paired Alignments with and without Normalization
The number of alignments is not reflective of the number of cells added tothe experiments targeting H3K4me3 (left panel) and YY1 (right panel), thus
normalization is needed.

CUT&RUN Spike-In| 9Spike-In Normalization: Complete Guide

Overview, Methods, and Resources

GSK126 global decrease in EED binding

Figure 6. Spike-In Normalization is Necessary to Reveal Global
Decrease in EED Binding in the Presence of GSK126 Treatment
500,000 K562 cells (Control) and 500,000 K562 cells treated with GSK126
were assayed in CUT&RUN for EED. Without Spike-In normalization, (top two
IGV tracks) no decrease in global EED binding is observed. However, with
Spike-In normalization (bottom two IGV tracks), a reduction in EED binding isobserved.

CUT&RUN Spike-In|

10

Spike-In Normalization: Complete Guide

Overview, Methods, and Resources

CUT&Tag-IT® Spike-In

How Does CUT&Tag-IT Spike-In Control Work?
Active Motif’s strategy for CUT&Tag normalization is to spikecryopreserved Drosophila cell nuclei into samples prior to CUT&Tag.
Then, during the primary antibody incubation step, a Drosophila H2Av
antibody is added in addition to the antibody targeting the histone markof interest. This Drosophila H2Av antibody does not cross react to otherspecies and provides a mechanism to reliably tag Drosophila chromatin
in a consistent way across all samples. A normalization factor is thencreated based on the Drosophila signal and applied to the test genome.
This CUT&Tag-IT® Spike-In Control strategy enables normalization of
CUT&Tag data independent of the experimental antibody and without bias.

CUT&Tag-IT® Spike-In Control Advantages⊲ Compare CUT&Tag datasets between samples⊲ Simply add Spike-In Nuclei to samples and perform
CUT&Tag with the Spike-In Antibody together
with the experimental target antibody⊲ Obtain normalization factor and reveal true
differences between samples

CUT&Tag-IT® Spike-In |11

Spike-In Normalization: Complete Guide

Overview, Methods, and Resources

Figure 7: CUT&Tag-IT Spike-In Control Method
Spike-In Nuclei are added to samples and assayed in CUT&Tag with the
Spike-In Antibody and experimental target antibody together in the same
reaction throughout the complete CUT&Tag assay.

| 12

Spike-In Normalization: Complete Guide

Overview, Methods, and Resources

CUT&Tag-IT® Spike-In Control Data

Figure 8: K562 CUT&Tag-IT Assay Results With andWithout CUT&Tag-IT Spike-In Control Normalization
IGV browswer tracks are shown for duplicate reactions of 50,000, 100,000,
150,000, and 200,000 K562 cells assayed in CUT&Tag for H3K27me3 without
spike-in and with CUT&Tag-IT Spike-In Control normalization. In the Spike-in
Normalized panel it is clear that the peak height correlates with increasingcell number, whereas in the No Spike-in control peak height differences are
not noticeable from 50,000 to 200,000 cells.

CUT&Tag-IT® Spike-In |13

Spike-In Normalization: Complete Guide

Overview, Methods, and Resources

H3K27me3 CUT&Tag

Figure 9. Spike-In Normalization After EZH2
Inhibition Reveals Expected Changes
IGV browser tracks are shown for control samples and samples treatedwith an EZH2 inhibitor. Data was analyzed both without and with Spike-in
normalization. Global decrease in H3K27me3 histone modification are only
detectable with Spike-in normalization.

CUT&Tag-IT® Spike-In |14

Spike-In Normalization: Complete Guide

Overview, Methods, and Resources

CUT&Tag-IT® R-loop Spike-In

Cleavage Under Targets and Tagmentation (CUT&Tag) has emerged asa powerful method for profiling the localization of histone modificationsof interest genome wide. Active Motif applied this approach to study
DNA-RNA Hybrid R-loops genome-wide with our CUT&Tag-IT® R-loop
Assay Kit. The CUT&Tag-IT® R-loop Assay Kit utilizes a CUT&Tag-based
approach to profile R-loops genome-wide using a specifically optimized
protocol for cell samples and our DNA-RNA hybrid (Clone S9.6) antibody.
Due to variation in cell numbers and NGS sequencing statistics between
samples, biological differences can be obscured. By applying Active
Motif’s CUT&Tag normalization strategy to the CUT&Tag-IT R-loop
Assay data, true differences between samples can be revealed.

CUT&Tag-IT® Spike-In Control, R-loop Advantages⊲ Compare CUT&Tag-IT R-loop Assay Kit
datasets between samples⊲ Simply add Spike-In Nuclei, R-loop to experimental samples
and perform CUT&Tag with the Spike-In Antibody, Mouse
together with the DNA-RNA Hybrid mAb (Clone S9.6)⊲ Obtain normalization factor and reveal true
differences between samples

CUT&Tag-IT® R-loop Spike-In | 15

Spike-In Normalization: Complete Guide

Overview, Methods, and Resources

Figure 11. Spike-In is Needed When Using Negative Control
RNase Treatment in CUT&Tag-IT R-loop Assay
R-loops are DNA-RNA Hybrids within the genome, and RNase A will cut
the hybrid, destroying the R-loop, thus RNase A treatment is used as anegative control in the CUT&Tag-IT R-loop Assay Kit. However, without
Spike-In Normalization, (top panel) no differences are observed. With Spike-In
Normalization, the effect of the treatment in the negative control is observed.

CUT&Tag-IT® R-loop Spike-In | 16

Spike-In Normalization: Complete Guide

Overview, Methods, and Resources

Resources & Ordering Information
Application Resource Centers
Whether you’re just beginning these techniques, or are interested inlearning more, access our complete resource centers for each. Get thelatest Webinars & Videos, Podcasts, eBooks, TechNotes & Protocols forthese and a range of epigenetic assay types:
ChIP-Seq

ATAC-Seq

CUT&RUN

CUT&Tag

DNA Methylation

Ordering Information
Name

FormatCat No.

Spike-in Antibody

50 µg

61686

Spike-in Chromatin

15 rxns53083

Drosophila Positive Control Primer Set Pbgs

96 rxns

71037

Drosophila Negative Control Primer Set 1

96 rxns71028

Drosophila Negative Control Primer Set 2

96 rxns71029

Drosophila Negative Control Primer Set 3

96 rxns

71038

ATAC-Seq Spike-In Control

16 rxns

53154

ATAC-Seq Kit

16 rxns

53150

Fixed Cell ATAC-Seq Kit

16 rxns

53151

CUT&RUN Spike-In Control

24 rxns

53183

ChIC/CUT&RUN Assay Kit

24 rxns

53180

ChIC/CUT&RUN pAG-MNase

50 rxns

53181

CUT&Tag-IT® Spike-In Control, Anti-Rabbit

16 rxns

53168

CUT&Tag-IT® Spike-In Control, Anti-Mouse

16 rxns

53173

CUT&Tag-IT® Assay Kit, Anti-Rabbit

16 rxns

53160

CUT&Tag-IT® Assay Kit, Anti-Mouse

16 rxns

53165

CUT&Tag-IT® Core Assay Kit

16 rxns

53176

CUT&Tag-IT® Spike-In Control, R-loop

16 rxns

53174

CUT&Tag-IT® R-loop Assay Kit

16 rxns

53167

Resources & Ordering Information |17

Spike-In Normalization: Complete Guide

Overview, Methods, and Resources

WebinarTo learn more, watch the Spike-In Normalization webinar atactivemotif.com/webinars
In this webinar, Brian Egan, Head of R&D at Active Motif discusses why
data normalization is often critical for sequencing-based assays like ChIPSeq, ATAC-Seq, CUT&RUN and CUT&Tag. The concept behind our suite of
Spike-In Controls is covered, as well as how and when to use these tools
to obtain the best possible results when analyzing experimental data.

| 18