Custom Services

Enabling Epigenetics Research

for Epigenetic and Gene Regulation

Custom Services

for Epigenetics and Gene Regulation Research

ChIP-Seq
End-to-End Services for genome-wide mapping of protein-DNA interactions
Chromatin immunoprecipitation (ChIP-Seq) combined with Next-Generation Sequencing is the most widely
utilized technique to study protein-DNA interactions and histone modification localization across the genome.
Given the importance of ChIP-Seq data sets for development and disease research, obtaining the highest
quality data is crucial.
Choose the Global Leader in End-to-End ChIP Services
Active Motif offers the most diversified portfolio of ChIP Services. We bring over a decade of experience
providing services, with over 10,000 samples processed, and the highest level of expertise of any
service provider.

Histone Mark ChIP-Seq

ChIP Antibody Validation

Transcription Factor ChIP-Seq

ChIP-Seq Spike-In Normalization

RNA Polymerase II ChIP-Seq

Super-enhancer ProfilingComprehensive
ChIP-Seq services
1

Customer fixes cell lines or
freezes tissue samples

2

Active Motif prepareschromatin and sonicates3

Active Motif performs the
ChIP reactions

4

Active Motif constructs
ChIP-Seq libraries

5

Libraries sequenced using
Illumina platform

6

Analysis and datadelivery

Quality control steps
to ensure success
Chromatin preparation isquantified and sized
Antibody is ChIP qualified
prior to ChIP-Seq ChIP rxns
Library is run on TapeStation
to confirm fragment sizes.
Library yield is quantified via
Qubit.
Sequencing to yield ~30
million tags

To learn more, visit activemotif.com/services-chipseq
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1

Custom Services

for Epigenetics and Gene Regulation Research

ChIP Antibody Validation Services
Services to test the suitability of your antibody for ChIP applications
One of the greatest challenges in ChIP experiments is the lack of available antibodies that can recognize
fixed, target-bound proteins and that function in immunoprecipitation. Active Motif’s ChIP Antibody
Validation Service makes this process simple, fast, and convenient.
Let the ChIP Experts® do the work for you.
Only 30% of all antibodies work in ChIP-Seq. Therefore, identification of a good ChIP-Seq antibody
presents a significant barrier to project initiation and completion. Our Epigenetic Services team has
validated antibodies to over 350 targets. If your target of interest is on our list, we can start your project
immediately. Otherwise, submit an antibody to us and our Antibody Validation Service can give you ananswer in as little as 4 weeks.

Hundreds of antibodies
already validated"Yes" or "No" results for
ChIP-Seq functionality

Submit any antibody for testing
To learn more, visit activemotif.com/ab-val

Super Enhancer Profiling
Specialized ChIP-Seq data generation and analysis services
for genome-wide super-enhancer profiling
Most genes that are considered master regulators are transcription factors. Super-enhancers are regulatory
regions that control the expression of these master transcription factors. Active Motif offers a specialized
ChIP-Seq service to identify super-enhancers which helps define the master regulators of any given cell
type or disease sample.

e1⊲ Identify master regulators ofcell identity

H3K4m

SOX2

OCT4

MED1

ac
H3K27

BRD4

There are many proteins that assemble into super-enhancers, however H3K27ac is a universal marker
of super-enhancers. Active Motif can generate a super-enhancer profile from any sample by simply
performing an H3K27ac ChIP-Seq experiment⊲ Find regulatory regions
associated with disease

To learn more, visit
activemotif.com/services-superenhancer
North America
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Europeeurotech@activemotif.com⊲ Determine mechanisms of
BRD4 inhibitors

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2Custom Services

for Epigenetics and Gene Regulation Research

ChIP-Seq Spike-In Normalization
A novel ChIP-Seq normalization strategy to reveal hidden biological effects
Active Motif's standard normalization method for ChIP-Seq data uses background signal for normalization,
allowing for differences in peak signals to easily be observed. However, standard ChIP-Seq normalization
may not be effective in some circumstances, for example when there are overall differences in sample
signals due to experimental effects. In these cases, using our spike-in chromatin and antibody isrecommended for normalization.

Uncover effects masked by large
differences in overall levels of
Immunoprecipitation

How Does It Work?
Experimental
Chromatin

Spike-In
Chromatin

Antibodyof Interest

Monitor consistency
between samples

Spike-In
Antibody

Reduce sample bias

Chromatin
Immunoprecipitation
Sequencing
Map to experimental
genomeSpike-in Normalization

DMSO

_

Inhibitor

DMSO+

Inhibitor

Map to Drosophila
genomeNormalize Drosophila
tag counts across samples

Figure 1.
Normalize experimental
sample tag counts
by same ratio

Why?
Without Spike-In normalization (-), unevenamplification of the ChIP DNA during
preparation of Next-Gen sequencing
libraries led to loss of differences between
samples. With Spike-In normalization (+)
the bias in PCR amplification was corrected
and the difference between samples isclearly visible.

Cells treated with a small molecule inhibitor of EZH2
methyltransferase have dramatic reductions in global
H3K27me3 levels. However, H3K27me3 ChIP-Seq using
standard ChIP-Seq protocols (-) does not detect thesedifferences. Incorporation of Active Motif’s ChIP-Seq SpikeIn Strategy (+) reveals the expected decrease in H3K27me3
ChIP-Seq signal.

To learn more, visit
activemotif.com/services-normalize* 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. 11:e0166438

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3Custom Services

for Epigenetics and Gene Regulation Research

CUT&Tag-IT®* Service
Tn5 Transposase assisted chromatin profiling

Cells

Target-Specific
Antibody

Secondary
Antibody

Protein A-Tn5

Cleavage Under Targets and Tagmentation(CUT&Tag) is a method to map genomic localizationof histone modifications that reveals interactions
between proteins and DNA or identifies DNA
binding sites for proteins of interest. CUT&Tag
utilizes antibody directed Tn5 Transposase
tagmentation* to target specific histone
modifications to create genome-wide maps. Tn5
tagmentation sharpens resolution and decreases
the sequencing depth requirement compared
to ChIP-Seq.

Best for histone
modification targets
Only low sequencing depth
requiredCompatible with a low
starting material

Mg++@37˚C

Low background signal
Figure 2.
Our CUT&Tag- IT®* Service is based on the same principles
as ChIP-Seq, but with several improvements advantageous
for mapping histone marks. In CUT&Tag, unfixed cells arebound to concanavalin A beads and the antibody incubationis performed with cells in their native state. Directly following
antibody binding, the chromatin is digested and NGS libraries
are prepared in a single step by tagmentation using the protein
A-Tn5 (pA-Tn5) transposase enzyme that has been pre-loadedwith sequencing adapters.

Extract DNA

Amplify
Sequencing
Library
Sequencing
and Analysis

To learn more, visit
activemotif.com/services-cut-and-tag* Our Tn5 Transposase mediated chromatin tagmentation methods are covered by these patents: US9938524, US10689643B2,
EP2783001B1, EP2999784B1.

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4Custom Services

for Epigenetics and Gene Regulation Research

ChIC/CUT&RUN Service
Native mapping of transcription factors and histone marks

Cells

Target-Specific
Antibody

Cleavage Under Targets & Release Using
Nuclease (CUT&RUN) is an epigenetic method
used to investigate the genome-wide distributionof various chromatin-associated proteins andtheir modifications. CUT&RUN is a derivative ofchromatin immunocleavage (ChIC). CUT&RUN issimilar to chromatin immunoprecipitation (ChIP),in that it utilizes an antibody to target chromatin
associated marks and proteins, but requires lesssample material and sequencing depths than ChIP.

Compatible with both histone markand transcription factor targets.
pAG-MNase

Requires fewer cells than ChIP
Low background signal.
Figure 3.

CaCl₂ @4˚C

In CUT&RUN, a protein of interest is tagged with an antibody
and bound to chromatin in intact cells. Then, a micrococcal
nuclease (MNase) is used to cleave the DNA specifically at thebinding sites of the protein of interest. 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.

ReleaseDNA @37º

NGS Library
Prep
Sequencing
Library
Sequencing
and Analysis

To learn more, visit
activemotif.com/services-cut-run
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5Custom Services

for Epigenetics and Gene Regulation Research

ATAC-Seq Services
Genome-wide identification of open chromatin regions
Assay for Transposase Accessible Chromatin Sequencing (ATAC-Seq) is designed to study open chromatin,
which is known to contain active gene regulatory elements including promoters, enhancers, and insulators.
This assay provides data to enable identification of accessible chromatin regions across the genome thatare distinct to individual cell types. ATAC-Seq is a perfect first step for those exploring the role of epigenetics
in cell systems or disease models for which little information is available on mechanisms of gene regulation.

Determine if Epigenetic Mechanisms Are at Work⊲ Gain mechanistic insight into gene regulation in response to treatment⊲ Identify which transcription factors are driving disease or response⊲ Generate genome-wide profiles from patient samples (cells or tissues)

Con rep1

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T1 + T2 rep1

T1 + T2 rep2

T1 + T2 rep3

⊲ Only 50,000 cells required

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Figure 4.

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T2 rep2

Active Motif’s ATAC-Seq assay was performed on control andtreated cells, each in triplicate. Hundreds of differential peaks
were detected. The one depicted is in an intergenic region.

0.87

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Figure 5.
Active Motif’s ATAC-Seq assay was performed under four
different cellular conditions, each condition in triplicate. The
Pearson correlation coefficients were generated and graphedfor each pair-wise comparison. The data demonstrates theassay is highly reproducible with correlation coefficients near
1 for replicates. Four separate groups are clearly visible in theheat map, showing that triplicates are more similar to eachother than to other samples and indicating differences between
sample types.

To learn more, visit
activemotif.com/services-atacseq
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6Custom Services

for Epigenetics and Gene Regulation Research

RRBS Services
Reduced Representation Bisulfite Sequencing
DNA methylation patterns are cell-type specific, and alterations in these patterns can be indicative ofdisease. RRBS is a bisulfite dependent method that provides single base pair resolution of cytosine
methylation at millions of locations and allowing for sample-to-sample comparisons of DNA methylation
patterns. Comparing DNA methylation profiles from normal and diseased patient samples can facilitate
novel biomarker discovery.
Why is RRBS the Right Choice?
RRBS is significantly less expensive than Whole Genome Bisulfite Sequencing, while still providing themethylation status of up to 5 million CpGs at biologically relevant positions such as promoters and CpG
islands. Send in cells, tissue, or purified DNA.

⊲ Low starting material requirements

Services Include⊲ Data provided on millions of CpGs⊲

Single base resolution⊲

Quantitation at each base⊲

Data at millions of locations acrossthe genome⊲

Data enriched at promoters and
CpG islands⊲

Dramatically less expensive thanwhole Genome Bisulfite Sequencing⊲ Data from biologically relevant regions⊲ Promoters⊲ CpG Islands

RRBS Service
Customers submit DNA, cell pellets or frozen tissue
then we perform:

1

DNA purification

2

DNA digestion

3

Sequencing adaptor ligation

4

Bisulfite conversion

5

PCR amplification

6

Sequencing on
Illumina platform

7

Bioinformatic analysis

Pie chart showing the assignment of differentially methylated
cytosines to genic features.
A differential methylation analysis was performed on a per CpG
basis, and identified sites were then aligned to annotated genefeatures such as promoters and exons.

To learn more, visit
activemotif.com/services-rrbs
North America
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Figure 6. RRBS Data from Human Samples

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Custom Services

for Epigenetics and Gene Regulation Research

Hi-C Services
Map Genome-wide chromatin-chromatin interactions using our Hi-C Service
Functional elements such as enhancers can influence gene expression by interacting directly withpromoters and other loci that may be thousands of kilobases away. Use our end-to-end Hi-C service
to map these interactions and get a 3D view of genome organization. Elements detected include A/B
compartments, topologically associated domains (TADs), and chromatin loops.

Schematic of Hi-C Workflow⊲ Identify changes inchromatin organizationin response to treatment
or stimuli
Start with cellor tissue samples

Crosslink
chromatin

Fragment
chromatin

Repair andbiotinylate ends

Ligate fragmented
chromatin

Shear DNAand pull down
biotinylated DNA

Paired-end
Sequencing

Bioinformatics⊲ Identify novel
chromatin organization
corresponding to different
stages of development.

Elements Detected Include:

Figure 7. Hi-C enables identification of complex chromatin interactions like chromatin looping and Topological Associating
Domains (TADs).
Hi-C was performed using mouse uterine tissue. Snapshot of Wash U Epigenome Browser looking at a 6.8 Mb region of chromosome
6. Topologically Associating Domains (TADs) are represented as purple bars. Chromatin loops are indicated by brown arcs.

To learn more, visit
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8Custom Services

for Epigenetics and Gene Regulation Research

Mod Spec® Services
Histone Modification Detection Service
Total nuclear levels of histone post-translational modifications (PTM) may differ under varying conditions –disease vs normal, DMSO vs inhibitor, or WT vs KO. Active Motif’s Mod Spec® service can verify expecteddifferences, and more importantly, identify unexpected changes in histone PTM levels. This service uses
mass spectrometry for relative quantitation of over 80 histone states.

Detect over 80 different histone states
Measure acetylation, methylation, ubiquitination, and unmodified peptides
Analyze histone modifications on H1, H2, H3.1, H3.3, and H4More quantitative and comprehensive than western blots or ELISAHow Does Modspec Work?
HistoneExtraction

Propionylation

Trypsin
Digestion

Mass Spec

Analysis

Figure 8. Mod Spec reveals the effectsof GSK-126 treatment on HeLa cells.
GSK126 is an inhibitor that blocks the
methyltransfease activity of EZH2,
resulting in global decreases in
H3K27me2 and H3K27me3. A selectionof H3 modifications are shown that
confirm significant decreases in H3K27
methylation with concomitant increases
in acetylated and unmodified H3K27(highlighted in yellow). Changes in
H3K36 are highlighted in red.

To learn more, visit
activemotif.com/modspec

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9Custom Services

for Epigenetics and Gene Regulation Research

Gene Expression Services
RNA-Seq for steady state mRNA levels
RNA Pol II ChIP-Seq for transcription rate measurements
Active Motif transcriptome analysis services include RNA-Seq for identification and quantitation of RNAtranscripts and RNA Pol II ChIP-Seq for quantitation of transcription rates to enable rapid profiling ofchanges in gene expression associated with transcription factor (TF) and histone modification occupancy.
RNA-Seq Services

RNA Pol II ChIP-Seq Services

Simply submit RNA, cells, or tissue samples. Order
RNA-Seq alone or combine with ChIP-Seq data to
uncover contextual information about:

Analysis of RNA Pol II occupancy as a proxymeasurement of transcription rates offers theadvantage of enabling you to:Differential gene expression
Changes in gene structure or
splicing patterns
Effects of TF binding on gene
expression

Measure transcription without the
influence of RNA half-life
Identify genes poised fortranscriptional activation
Generate gene expression datafrom cells used for ChIP-Seq
Measure changes at early time
points post treatment1

Prepare RNA from cells or
tissues

2

Generate cDNA

3Construct directional
libraries

4

Perform Next-Generation
Sequencing

5

Perform data analysis

RNA Pol II Changes Are Detected Earlier Than mRNA Changes
6

RNA Pol II ChIP-Seq
RNA-Seq

Fold Induction

5
4
3
2
1
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100 120 140 160 180 200

MinutesFigure 9: Gene expression profiles vary depending on theanalysis method.
Data for Igf1r was extracted from RNA–Seq and RNA Pol II
ChIP-Seq data sets. Cell treatment resulted in induced gene
expression that was measured at various time points. The
cumulative data show that transcription, as measured by RNA
Pol II ChIP-Seq, is induced immediately, while mRNA levels only
accumulate over time.

To learn more, visit
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10Custom Services

for Epigenetics and Gene Regulation ResearchInteractome Profiling (RIME)
Mass Spectrometry identifies co-factor recruitment into transcriptional complexes
RIME (Rapid Immunoprecipitation Mass Spectrometry of Endogenous Proteins) sheds light on the complex
process of gene regulation by enabling capture and identification of chromatin associated proteins thatinteract with an endogenous protein of interest.
Why RIME?
Gene regulation is often oversimplified when thefocus is on one particular transcription factor inany given cell model. In reality, differential gene
expression is greatly influenced by co-factors andother protein interactions within chromatin. RIME
clarifies this complexity by providing a means to
identify the protein interactions that are importantfor gene regulation.

Enables capture of low affinity interactions
Allows more stringent wash conditions
resulting in less non-specific interactions

How Does RIME Work?

Experimental Design⊲

Antibody validation is performed on asingle sample to show that the target
protein is detected⊲

IP-mass spec using the target antibody
is performed in duplicate⊲

IP-mass spec using anti-lgG is
performed in duplicate⊲

Targets DNA/chromatin associatedproteins

IgG interactions are removed from thetarget antibody specific interaction list

Targeted
TF
Targeted
TF
DNA binding direct interactor

DNA binding direct interactor

Non-DNA binding direct interactor

Non-DNA binding direct interactor

Indirect interactor

Indirect interactor

Figure 10. Example data from RIME
Different Estrogen Receptor binding profiles have been observed depending on the ligand used to stimulate binding. Differences are
known to occur through ligand induced conformational changes in the receptor that influence cofactor recruitment. Our RIME datashows differential recruitment of cofactors to DNA bound estrogen receptor after treatment with ligand 1 and ligand 2. Light purple
indicates recruited proteins with similar rank order for both ligands. Tan indicates common protein detected but with different rankorder. Dark Purple indicates unique protein recruitment.

Ligand 1

Ligand 2

Estrogen Receptor

Estrogen Receptor

Nuclear receptor co-activator 3

Vang-like protein 1

Nuclear receptor interacting protein 1

Pericentriolar material 1 protein

Pericentriolar material 1 protein

Centrosomal protein of 131 kDa

Centrosomal protein of 131 kDa

Protein GREB1

CREB-binding protein

E3 ubiquitin-protein ligase TRIM33

E3 ubiquitin-protein ligase TRIM33

Nuclear receptor interacting protein 1

To learn more, visit activemotif.com/rime

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11Custom Services

for Epigenetics and Gene Regulation ResearchSingle-Cell RNA-Seq Services
Measure gene expression in heterogeneous populations at single-cell resolution
Single-Cell RNA-Seq enables transcriptome analysis at the single-cell level. scRNA-Seq can be used to
identify cell subpopulations with different transcriptome profiles within complex samples, eliminating theneed for isolation strategies like FACS or magnetic sorting that could alter the biology of the sample due tosample manipulation.
To learn more, visit activemotif.com/services-scrna-seq
Figure 11. Identify unique subpopulationsof cells within a single sample.

2_4428AM_PBMC-B_scRNA_hg38
2_4428AM_PBMC−B_scRNA_hg38
Cluster UMAP
Cluster UMAP

Single-Cell RNA-Seq data generated fromhuman PBMCs. Each color-coded clusteron the UMAP plot represents populationsof cells that have the same gene
expression profile. 20 refined clusters
were identified.

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Active Motif’s End-to-End
scRNA-Seq service includes:⊲

Cell preparation⊲

Sample processed using 10X
Genomics Chromium platform⊲

Library generation⊲

Sequencing⊲

Bioinformatic analysis

−15
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−10

−5

UMAP−10

510Single-Cell ATAC-Seq Services
End-to-End service to identify open chromatin regions at single-cell resolution
Active Motif’s scATAC-Seq service enables examination of genome-wide chromatin accessibility of
thousands of cells in parallel, allowing examination of subpopulations of cells within a heterogeneous
population that would otherwise be lost in standard bulk ATAC-Seq.
To learn more, visit activemotif.com/services-scmultiome
Cell Clustering (By Cluster)
Cell Clustering
Sample mKidney- (By Cluster)
Sample mKidney​​​​

Cluster 1 (1204)

40

40

Cluster 2 (850)
Cluster 3 (600)30tSNE axis 2

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tSNE axis 2

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Cluster 4 (593)20

Cluster 5 (503)10Cluster 7 (474)0Cluster 9 (396)−10Cluster 11 (335)

Cluster 6 (476)
Cluster 8 (451)
Cluster 10 (361)
Cluster 12 (211)

Cluster 1 (1204)
Cluster 2 (850)
Cluster 3 (600)
Cluster 4 (593)
Cluster 5 (503)
Cluster 6 (476)
Cluster 7 (474)−20

Cluster 8 (451)−30

Cluster 9 (396)
Cluster 10 (361)

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−40

Identify variations in chromatin accessibility across different cell populations within a single sample.
−40

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0

North AmericatSNE axis 1
tech_service@activemotif.com

20

Active Motif’s End-to-End
scATAC-Seq service includes:⊲

Cell preparation⊲

Transposase reaction⊲

Sample processed using 10X
Genomics Chromium platform⊲

Library generation⊲

Sequencing⊲

Bioinformatic analysis

40

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12Custom Services

for Epigenetics and Gene Regulation ResearchSingle-Cell Multiome Service
End-to-End service to measure gene expression andopen chromatin states from the same cell
Single-Cell Multiome allows for both transcriptome analysis and genome-wide detection of open
chromatin at the single cell level. Understanding both the gene expression profile and the chromatin state
at single-cell resolution can help identify how epigenetic changes instruct gene expression in distinct
cell populations.

GEM
Beads

Bulk ATAC
Reaction

Tn5 Tagmented
Nuclei

Oil in Well

What are the advantages ofusing Single-Cell Multiome?
Single-Cell Multiome can be used
to identify cell subpopulations
with different transcriptomal
and epigenetic profiles withincomplex samples, eliminating theneed for isolation strategies like
FACS or magnetic sorting that
could alter the biology due tosample manipulation.
For example:

Collect⊲

Identifying novel cell
subpopulations that modulate
response to drug treatments(e.g., responders vs.
resistant cells)⊲

Identifying subpopulationsof cells with variations
in gene expression thatcan provide insight intodevelopmental trajectories(e.g., brain development,
T-helper cell development,
B-cell differentiation)

Single Nuclei
GEMs

RT and Barcode Attachment

Barcoded mRNA andATAC DNA

Pre-amplificationscATAC-Seq LibraryscRNA-Seq Library

Figure 13.
Single-Cell Multiome measures gene expression and open chromatin from the samecell. Tn5 tagmentation is performed on nuclei, which are loaded onto the 10X Genomics
Chromium Controller and met with GEMs (gel bead-in-emulsion) containing reverse
transcriptase and sequencing adapters. Open chromatin fragments and cDNAs arebarcoded, creating two unique libraries per cell.

Active Motif’s End-to-End
scRNA-Seq service includes:⊲

Cell preparation⊲

Sample processed using 10X
Genomics Chromium platform⊲

Library generation⊲

Sequencing⊲

Bioinformatic analysis

To learn more, visit activemotif.com/services-scmultiome
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Custom Services

for Epigenetics and Gene Regulation Research

Bioinformatics
Comprehensive and customizable data analysis and support
from our expert team of scientists
At Active Motif, our team of bioinformatic scientists has been providing premium bioinformatic analysis aspart of our end-to-end Epigenetic Services for more than a decade. Now, we're offering this expertise forour customers who just require assistance with their bioinformatics. Perhaps you sent your samples away
for sequencing and now don’t know where to begin with your delivered sequencing files. Aren’t sure how
to move forward? We can help.

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Custom Services
The Active Motif Custom Services team makes cutting-edge research accessible to the wider
life science community. We provide services for state-of-the-art epigenetics and gene regulation
analysis techniques to accelerate your research.

ChIP-Seq

IP-Mass Spec

CUT&Tag

RIME

ChIC/CUT&RUN

Single-cell ATAC-Seq

ATAC-Seq

Single-cell/nucleus RNA-Seq

RRBS

Single-cell Multiome

RNA-Seq

Hi-C

Histone Modification Analysis

Bioinformatics

North America
Toll Free:
Direct:Fax:
E-mail:Europe877 222 9543
760 431 1263
760 431 1351
tech_service@activemotif.com

Japan

Direct: +32 (0)2 653 0001
Fax: +32 (0)2 653 0050
E-mail: eurotech@activemotif.com

China
Direct: +81 (0)3 5225 3638
Fax: +81 (0)3 5261 8733
E-mail: japantech@activemotif.com

Direct: +86 21 20926090
Cell Phone: 18521362870
E-mail: techchina@activemotif.com