ChIP-Bis-Seq Kit(version A1)

Catalog No. 53048

Active Motif North America
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Information in this manual is subject to change without notice and does not constitute a commitment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any warranty of any
kind, either explicit or implied. Information may be changed or updated in this manual at any time.
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© 2014 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA 92008. All rights reserved.
All trademarks, trade names, service marks or logos referenced herein belong to their respective
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TABLE OF CONTENTS

Page

Overview  . . . . . . .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Flow Chart of Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Introduction  . . . .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
References . . . . . .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4
Kit Performance and Benefits  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Protocol Overview and Time Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Kit Components and Storage .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 7Module 1: ChIP-IT® High Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Module 2: Sequencing Library Preparation  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Module 3: Bisulfite Conversion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  10
Protocols – Chromatin Preparation and Immunoprecipitation .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 11
Cell Growth and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Reagent Information  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section A. Cell Fixation Starting with Cultured Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Section B. Chromatin Sonication of Cultured Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  16
Section C. Cell Fixation Starting with Fresh or Frozen Tissue . . . . . . . . . . . . . . . . . . . . . . .  18
Section D. Chromatin Sonication of Tissue  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  19
Section E. Immunoprecipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Section F. Reversal of Cross-links and DNA Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Protocols – Sequencing Library Preparation . .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 24
Reagent Information  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Section G. Perform End Repair  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Section H. Add ‘A’ Overhang to 3’ end of DNA Fragments . . . . . . . . . . . . . . . . . . . . . . . . . 25
Section I. Ligate Adapters to DNA Fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Protocols – Bisulfite Conversion .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 26
Reagent Information  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Section J. Bisulfite Conversion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Section K. On-column Desulfonation and Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Section L. Enrich the Adapter-Modified DNA Fragments by PCR . . . . . . . . . . . . . . . . . . . 28
Section M. Size Select the Library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Section NJ. Analyzing the Sequencing Results  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Appendix
Section O. Adapter and Primer Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Section P. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Section Q. Related Products  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Technical Services  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

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Overview
Active Motif’s ChIP-Bisulfite-Sequencing (ChIP-Bis-Seq) Kit combines the target-specific selectionof chromatin immunoprecipitation (ChIP) with bisulfite conversion and sequencing (Bis-Seq)
to provide single-nucleotide resolution of immunoprecipitated DNA. ChIP is a powerful tool forstudying protein/DNA interactions because it identifies the localization of proteins bound to
specific DNA loci. While bisulfite conversion and subsequent DNA sequencing is the gold standard
for DNA methylation analysis because it provides detailed information on the methylation patternof individual DNA molecules at single-base-pair resolution. When the two techniques are used in
combination, it generates multi-parametric information about individual DNA molecules enabling
better understanding of the biological context and significance regarding gene regulation, gene
expression, mechanisms of chromatin modification and pathway analysis.
The ChIP-Bis-Seq Kit consists of three modules. The first is Active Motif’s ChIP-IT High Sensitivity
Kit which is used to immunoprecipitate chromatin bound to a protein of interest in order to identify DNA binding sequences. Using our ChIP-IT High Sensitivity assay, chromatin is prepared from
cells or tissues, sonicated to fragment the chromatin and up to 16 chromatin immunoprecipitation reactions are performed. The second kit module contains methylated adapters and enzymes
necessary to prepare 10 next generation sequencing (NGS) libraries from the ChIP DNA. The third
kit module contains reagents to perform bisulfite conversion on the adapter-ligated DNA to determine the methylation status of each DNA strand. For successful ChIP-Bis-Seq, we recommend
using at least 30 µg chromatin (or 4.5 million cell equivalents) per ChIP reaction.

product

formatcatalog no.

ChIP-Bis-Seq Kit

10 libraries

53048

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1Flow Chart of Process

Chromatin Preparation

Chromatin
Immunoprecipitation

DNA Purification

Sequencing Library Construction• Perform End Repair• Add ‘A’ Overhangs to 3´ Ends
• Ligate Methylated Adapters

Bisulfite Conversion
Me

Me

Me

GATCGACGATCGGAGCGTAGGTACGACGTT
Bisulfite Conversion

GATCGAUGATCGGAGUGTAGGTACGAUGTTPCR Amplification

GATCGATGATCGGAGTGTAGGTACGATGTTPCR Amplification
Size Select the
Library

Sequencing

Flow chart of the ChIP-Bisulfite-Sequencing Method.
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Introduction
DNA methylation is an essential epigenetic modification that plays a key role in transcriptional
regulation and assures the proper regulation of gene expression and stable gene silencing innormal cells. DNA methylation is the covalent addition of a methyl group to the fifth positionof the cytosine pyrimidine ring in DNA and usually occurs within CpG dinucleotides, although insome cases the DNA methylation can also be found in a non-CpG context1, 2. CpG dinucleotides
are concentrated in large clusters called CpG islands, which are often positioned in the promoter
regions of many genes and are usually unmethylated3. It is estimated that the human genome
contains around 29,000 CpG islands and that almost 60% of all human genes are associated with
CpG islands4. Aberrant cytosine methylation is associated with silencing of tumor suppressor
genes5 and plays a decisive role in the development of many cancers6-14. Alterations in the methylation status of DNA are promising candidates for highly specific and sensitive indicators of cancer
diagnosis and prognosis15-17. Apart from carcinogenesis, DNA methylation is crucial for a variety ofprocesses, such as genomic imprinting, X-chromosome inactivation, and suppression of repetitiveelements4, 18. Thus profiling DNA methylation across the genome is vital to understanding the influence of epigenetics.
The bisulfite reaction was first described in the early 1970s19 and was used by Frommer et al.20,
and Clark et al.21 to distinguish between cytosine and 5-methylcytosine (5-mC) in DNA. It is now
known that both 5-methylcytosine and 5-hydroxymethylcytosine (5-hmC) remain unchanged
during conversion and therefore this method cannot be used to distinguish between the different
methyl modifications22-24. In the bisulfite conversion reaction, DNA is first treated with sodium
bisulfite to convert cytosine residues into uracil in denatured (single-stranded) DNA, underconditions whereby 5-mC and 5-hmC remain essentially non-reactive. The DNA is then amplified
by PCR where the uracils are converted to thymines. Bisulfite converted DNA can be analyzed
for gene- or allele-specific methylation patterns, or adapted for genome-wide DNA methylation
analysis. A methylation profile of the sample can then be created by comparing the sequence ofthe converted DNA to untreated DNA.
Me

Me

Me

GATCGACGATCGGAGCGTAGGTACGACGTT
Bisulfite Conversion

GATCGAUGATCGGAGUGTAGGTACGAUGTTPCR Amplification

GATCGATGATCGGAGTGTAGGTACGATGTT

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References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.

Ramsahoye, B.H. et al. (2000) Proc. Natl Acad. Sci. USA, 97: 5237-5242.
Lister, R. et al. (2009) Nature, 462: 315-322.
Antequera, F. and Bird, A. (1993) Proc. Natl Acad. Sci. USA, 90: 11995-11999.
Bird, A. (2002) Genes Dev, 16: 6-21.
Issa, J.P. (2004) Nat Rev Cancer, 4: 988-993.
Toyota M., et al. (1999) Proc. Natl Acad. Sci. USA, 96: 8681-8686.
Hughes, L.A. et al. (2012) Biochim Biophys Acta., 1825: 77-85.
Samowitz, W.S. (2007) Mol Diagn., 9: 281-283.
Marsit, C.J. et al. (2006) Cancer Res., 66: 10621-10629.
Suzuki, M. et al. (2006) Cancer, 106: 2200-2207.
Shen, L. et al. (2002) J Natl Cancer Inst., 94: 755-761.
Fang, F. et al. (2011) Sci Transl Med., 3:75ra25.
Jones, P.A. and Baylin, S.B. (2002) Nature Rev. Genet., 3: 415-428.
Esteller, M. et al. (2001) Cancer Res., 61: 3225-3229.
Laird, P.W. (2003) Nat Rev Cancer, 3: 253-266.
Van Engeland, M. et al. (2011) J Clin Oncol., 29: 1382-1391.
Esteller, M. (2008) N Engl J Med., 358: 1148-1159.
Reik, W. et al. (2001) Science, 293: 1089-1093.
Shapiro, R. et al. (1974) J Am Chem Soc, 96(3): 206-212.
Frommer, M. et al. (1992) Proc. Natl Acad. Sci. USA, 89: 1827-1831.
Clark, S.J. et al. (1994) Nuc. Acids Res., 22: 2990-2997.
Huang, Y. et al. (2010) PLoS One, 26: e8888.
Jun, S. et al. (2010) Nucl. Acids Res., 38: e125.
Nestor, C. et al. (2010) BioTechniques, 48: 317-319.

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4Kit Performance and Benefits
The ChIP-Bis-Seq Kit is for research use only. Not for use in diagnostic procedures.
PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING!
ChIP Bisulfite Sequencing Advantages:• Directly interrogate both chromatin and DNA methylation profiles on the same sample withhigher resolution than either ChIP or bisulfite sequencing alone• Evaluate allele-specific difference in DNA (imprinting, X-inactivation)• Method works with histone and transcription factor ChIP antibodies• Optimized ChIP reagents reduce background levels caused by non-specific binding events
providing high quality DNA for analysis• Reduce sequencing costs by focusing on specific genomic regions

Protocol Overview and Time Table
The ChIP-Bis-Seq Kit requires multiple days to complete. It is strongly advised to read the entire
protocol before starting and to plan your experiments in advance.
Required Time
Cell or Tissue Fixation and Lysis

1.5 hours

Chromatin Sonication

20 minutes per sample

Assessment of Chromatin Size*

4.5 hours for cell culture
Overnight for tissue

Immunoprecipitation

Overnight incubation

Binding to Protein G agarose Beads

3 hours

Wash Immune Complexes

20 minutes

Reversal of Cross-links

2.5 hours

ChIP DNA Purification

15 minutes(Optional) qPCR Analysis of ChIP enriched DNA

2 hours

Perform End Repair

45 minutes

Add ‘A’ Overhang to 3´ Ends of DNA fragments

45 minutes

Ligate Methylated Adapters to DNA Fragments

30 minutes

Bisulfite Conversion

5.5 hours

PCR Amplify Adapter-Modified Library

1 hourSize Select the Library

1.5 hours* The protocol varies between cell culture and tissue samples.

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Figure 1: ChIP-Bis-Seq sequencing and DNA methylation results.
This figure shows an example of sequencing results using the ChIP-Bis-Seq Kit to analyze the DNA methylation profile of
DNA that was enriched using a ChIP antibody for Histone H3K27me3 in PC9 cells. The top track shows a 423 bp region ofchromosome 7 from Human Hg19 genome. The next track reveals the ChIP-seq peak profile for Histone H3K27me3. The
final track shows the DNA methylation profile of forward and reverse reads as analyzed by bisulfite conversion of the ChIPenriched DNA. The blue bars represent unmethylated CpGs, while the red bars represent CpGs containing methylation. (Fora color image, please visit our website at www.activemotif.com/chipbisseq to download a color pdf of the manual).

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6

Kit Components and Storage
Module 1: ChIP-IT High Sensitivity (16 ChIP reactions)
Please store each component at the temperature indicated in the table below. Do not re-freeze
the Protein G Agarose Beads. Once thawed, Protein G beads should be stored at 4°C.
Reagents

Quantity

Storage

RNase A (10 µg/µl)

40 µl

-20°C

Proteinase K (10 µg/µl)

200 µl-20°C

Blocker

100 µl-20°C

100 mM PMSF

500 µl-20°C

Protease Inhibitor Cocktail (PIC)

500 µl-20°C

Precipitation Buffer

1.5 ml-20°C

Carrier

35 µl

-20°C

10X PBS

120 ml-20°C

Fixation Buffer

2 x 1.5 ml

4°C

Protein G Agarose Beads*

500 µl4°C

Stop Solution

20 ml

RT

Chromatin Prep Buffer

85 ml

RT

ChIP Filtration Columns

16 ea

RT

ChIP Buffer

35 ml

RT

5 M NaCl

400 µlRT

TE pH 8.0

2 x 1.5 ml

RTDetergent

25 ml

RT

Wash Buffer AM1

100 mlRT

Elution Buffer AM4

2 x 1.5 ml

RT

DNA Purification Binding Buffer

50 ml

RT

3 M Sodium Acetate

500 µlRT

DNA Purification Wash Buffer**

10 ml

RT

DNA Purification Elution Buffer

5 ml

RT

DNA Purification Columns

16 ea

RT

* The Protein G Agarose Beads are shipped on dry ice and can be stored frozen until their first use.
Once thawed, the Protein G beads should not be re-frozen by the customer, but stored at 4ºC.
**Requires the addition of ethanol before use.
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7

Additional materials required• A ChIP-validated antibody directed against the protein of interest• Dounce homogenizer with a small clearance pestle (e.g. Active Motif Catalog Nos. 40401& 40415) with the tight-fitting “A” pestle). Use of a homogenizer is necessary for shearing
chromatin.
• 37% formaldehyde solution with 10-15% methyl alcohol to prevent polymerization. Do not
use paraformaldehyde.
• For tissue preparations you will need Phenol and chloroform/isoamyl alcohol (24:1) (DNAPurification, Molecular Biology Grade)• 100% ethanol (absolute)• 70% ethanol• DNase-free H2O• Rocking platform for culture plates• Apparatus to rotate tubes end-to-end at 4°C (e.g. a Labquake from Barnstead/Thermolyne
with a tube holder for 1.5 ml microcentrifuge tubes)• Microcentrifuge (table top centrifuge 4°C) and microcentrifuge tubes• 250 µl PCR tubes• Thermocycler• 15 and 50 ml conical tubes• Spectrophotometer for DNA quantitation• Pipettors and tips (filter tips are recommended)• Sonicator (e.g. Active Motif’s EpiShear™ Sonicator with a 1/8” probe (Catalog No. 53051) with
the EpiShear™ Cooled Sonication Platform (Catalog No. 53080))• Agarose gel electrophoresis apparatus• Razor blades (for tissue preparations)• Hand-held homogenizer for tissue preparations (e.g. Biospec Products Tissue-Tearor)• Cell scraper (rubber policeman)• (Optional) ChIP-IT® qPCR Analysis Kit (Catalog No. 53029)• (Optional) Gene-specific qPCR primer pairs for enrichment analysis; see Appendix Section I• (Optional) SYBR Green qPCR master mix (Bio-Rad Catalog No. 170-8882)

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8Module 2: Sequencing Library Preparation (10 libraries)
Sequencing Library components arrive on dry ice and should be stored at -20ºC. Components areguaranteed stable for 6 months from date of receipt when stored properly.
Reagents

Quantity

Storage

DNA Polymerase I Klenow (5 U/µl)

5 µl

-20°C

T4 DNA polymerase (3 U/µl)

10 µl

-20°C

T4 Polynucleotide Kinase (10 U/µl)

10 µl

-20°C

Klenow Fragment (3’-5’ exo-) (5 U/µl)

10 µl

-20°C

10X Reaction Buffer AM3

50 µl

-20°C

T4 DNA Ligase (2,000 U/µl)

10 µl

-20°C

10X T4 DNA Ligase Buffer

50 µl

-20°C

2X Quick Ligation Buffer

2 x150 µl-20°C

dNTP Mix, 10 mM each

35 µl

-20°C

1 mM dATP

100 µl-20°C

Methylated Adapters (5 µM)

30 µl

-20°C

Glycogen (for use with Module 3)

10 µl

-20°C

Additional materials required• Purified ChIP DNA for each sample to be tested• QIAquick PCR Purification Kit (QIAGEN Cat. No. 28104)• MinElute PCR Purification Kit (QIAGEN Cat. No. 28004)• 250 µl PCR tubes• Thermocycler

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9Module 3: Bisulfite Conversion (10 conversion reactions)
Bisulfite Conversion components arrive at room temperature and can be stored at 4ºC prior to
first use. Then, we recommend storing each component at the temperatures listed in the table below. Components are guaranteed stable for 6 months from date of receipt when stored properly.
Reagents

Quantity

Storage

Conversion Reagent

2 tubes

RT

Hydroquinone

2 tubesRT

Buffer A

1.5 mlRT

Buffer B

1 ml

RT

DNA Binding Buffer

15 ml

RT

DNA Wash Buffer

20 ml

RT

DNA Elution Buffer

2.5 mlRT

ChIP-Bis-Seq purification columns

10 ea

RT

Additional materials required• Purified adapter-ligated sample DNA• 0.2 ml PCR tubes and caps
• 100% isopropanol• Microcentrifuge tubes and microcentrifuge• Thermocycler• Hot Start Taq polymerase and PCR reaction buffer recommended for use with bisulfite converted DNA (e.g. KAPA HiFi HotStart Uracil+ Ready Mix, KAPA Biosystems Catalog No. KK2801• PCR primers for library amplification (see recommendations in Appendix X)• 1.5% agarose TAE gel• Gel apparatus and power supply• 6x Loading dye• Molecular weight marker• QIAquick Gel Extraction Kit (QIAGEN Cat. No. 28704)• Razor blades (for gel extraction)• Picogreen or equivalent fluorescent quantitation method to determine DNA concentrationwww.activemotif.com

10

Protocols
Module 1 – ChIP-IT® High Sensitivity
The first step of the assay is to perform chromatin immunoprecipitation using the ChIP-IT®
High Sensitivity Kit module. Please refer to pages 7-8 of the manual for the kit components andadditional materials required to complete the chromatin preparation and immunoprecipitation
protocols.

Cell Growth Recommendations
When planning an experiment, calculate the number of chromatin preparations you will requireand determine the number of ChIP reactions you plan to perform on each chromatin preparation.
Be sure to include the appropriate positive and negative control ChIP reactions in your calculations. Also, note that if you wish to analyze the effect of particular compounds or culturingconditions on transcription factor/DNA interactions, you should prepare chromatin from control(untreated) cells as a reference sample.
The minimum recommended number of cells that should be used for the preparation of chromatin is 4.5 million cells per sample. It is then recommended to use 30 µg chromatin for each ChIP
reaction to ensure a minimum of 10 ng DNA for downstream bisulfite conversion. The quantityof the ChIP enrichment will depend on the abundance of the protein of interest and the affinityof the ChIP antibody. For highly abundant histone proteins, less chromatin may be used. For low
abundance transcription factors, it may be necessary to set up multiple ChIP reactions and combine the samples onto a single DNA purification column during the DNA purification and elution in
Section F, step 3
60 mm
dish

100 mmdish

150 mmdish

Seeding Density

0.8 x 106

2.2 x 106

5.0 x 106

Cells at 70-80%
Confluency*

2.4 x 106

6.6 x 106

15.0 x 106

Growth Medium
Volume

5 ml

10 ml

20 ml

Cell Fixative Solution

500 µl

1 ml

2 ml

Stop Solution

275 µl

550 µl1.1 mlPBS Wash Buffer(used per wash)

2 ml

5 ml

10 ml

Chromatin Prep Buffer

2 ml

5 ml

5 ml

ChIP Buffer

500 µl500 µl500 µl* The number of cells on a confluent plate or dish will vary with cell type. For this table, HeLa
cells were used. Please adjust as needed based on your particular cell type.
**Please refer to the descriptions below for complete details on buffer preparations
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Reagent Information
Complete Cell Fixation Solution
Buffer should be prepared fresh before each experiment. For every 20 ml of cell growth medium
used, prepare 2.5 ml of Complete Cell Fixation Solution by adding 180 µl Fixation Buffer to 1.57 ml
sterile water in a 15 ml conical tube. Using appropriate precautions (i.e. safety glasses, gloves andlab coat), add 750 µl 37% formaldehyde to the tube and vortex to mix. Use 1/10 growth medium
volume per plate.
Complete Tissue Fixation Solution
Buffer should be prepared fresh before each experiment. Prepare 10 ml of Tissue Fixation
Solution for each tissue sample to be processed by adding 1 ml 10X PBS to 8.7 ml sterile water in a
15 ml conical tube. Using appropriate precautions (i.e. safety glasses, gloves and lab coat), add
280 µl 37% formaldehyde to the tube and vortex to mix.
Stop Solution
Is provided ready to use. Use 1/20 media volume per cell culture plate or 515 µl per 10 ml Complete
Tissue Fixation Solution.
PBS Wash Buffer
Prepare 25 ml PBS Wash Buffer for every 15 cm plate or tissue sample. To a 50 ml conical tube add
21.25 ml sterile water, 2.5 ml 10X PBS and 1.25 ml Detergent. Mix by inverting. Place PBS Wash Buffer
on ice to chill. PBS Wash Buffer can be prepared in large quantities and stored at 4ºC for 6 months.
100 mM PMSF and Protease Inhibitor Cocktail (PIC)
Thaw the PMSF and the PIC at room temperature until fully dissolved, which takes about
30 minutes. Vortex gently and spin down briefly before use, then add to the buffers immediately
before use.
Chromatin Prep Buffer
Is supplied ready to use.
ChIP Buffer
Is supplied ready to use.
Protein G Agarose Beads
The supplied agarose beads require washing prior to use. Follow the instructions in the manual towash the beads for use in the ChIP reactions. There is no need to pre-block the beads or pre-clear
the sample. For best results, gently shake and invert the tube to resuspend the agarose beads. The
beads settle quickly, and therefore should be resuspended just before pipetting. We recommend
cutting 2 mm from the end of a pipet tip prior to pipetting to prevent the tip from becoming
clogged. Protein G Agarose Beads are shipped on dry ice, but should not be re-frozen by thecustomer. The beads should be stored at 4ºC.

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12

DNA Purification Wash Buffer
The DNA Purification Wash Buffer requires the addition of ethanol before use. The final concentration of ethanol should be 80%. Add 40 ml of fresh 100% ethanol to the DNA Purification Wash
Buffer bottle. Invert repeatedly. The buffer can be stored at room temperature after the additionof ethanol. The ethanol only needs to be added before the first use, after that the Wash Buffer isready for use.
3M Sodium Acetate
It is important to check the sodium acetate before use to ensure that the salts have not precipitated out of solution. Once the sodium acetate is in solution it should be stored at room
temperature.

Recommendations
ChIP-validated Antibody
We recommend using 4 µg antibody per ChIP reaction in a maximum volume of 30 µl. However,this will vary according to the affinity of the antibody and the quality of the chromatin; you may
need to use more of a particular antibody. ChIP antibodies must recognize fixed, native proteinthat is bound to DNA and/or complexed with other proteins. Many antibodies that perform wellin other applications do not perform in ChIP. Thus, ChIP performed with an antibody that has notbeen ChIP-validated must include appropriate controls (such as Active Motif’s ChIP-IT Control
qPCR Kits, Catalog Nos. 53026, 53027 and 53028) to validate the chromatin preparation and theChIP methodology. To see a list of available ChIP-validated antibodies available from Active Motif,
please visit www.activemotif.com/chipabs.
Chromatin Shearing Tips
ChIP experiments usually require chromatin that has been sheared to a size of 200-1200 bp. In
general, shearing efficiency is improved through the use of a small shearing volume and a V-bottom
tube rather than a round-bottom tube. Also, note that shearing is inefficient if the chromatin
sample becomes emulsified with air bubbles. To determine the appropriate shearing level for yoursample, set up a “practice” tube containing only ChIP Buffer. Slowly increase the sonication amplitude until foaming starts to occur. Reduce the amplitude setting down slightly and mark this as thehighest possible intensity to use without foaming. If a chromatin preparation becomes emulsified
inadvertently, discontinue shearing and centrifuge the sample at maximum speed for 4 minutes at
4ºC in a microcentrifuge to remove trapped air. Finally, to prevent overheating and denaturation ofchromatin, samples should be kept on ice as much as possible during shearing, and shearing shouldbe performed discontinuously (i.e. sonicate for 20 seconds, then place on ice/water for 30 seconds,
sonicate again for 20 seconds, etc.). If possible, shear while on ice or use Active Motif’s EpiShear
Cooled Sonication Platform (Catalog No. 53080) to help regulate sample temperature.

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13

Chromatin Quantity
It is recommended to use 30 µg chromatin per IP reaction (4.5 million cell equivalents). However, if
chromatin is in limited supply and the target protein is a highly abundant histone, lower chromatinamounts (10 µg or 1.5 million cell equivalents) may be used due to the high quality of the DNA
obtained from the ChIP-IT Bisulfite Sequencing Kit.
Safety Precautions
Formaldehyde and PMSF are highly toxic chemicals. Appropriate safety precautions (i.e. safety
glasses, gloves and lab coat) should be used. Also, formaldehyde is highly toxic by inhalation andshould be used only in a ventilated hood. Finally, chromatin sonication should be performed in abiosafety hood if the chromatin is extracted from biohazardous or infectious materials.

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14Section A: Cell Fixation Starting with Cultured Cells
This protocol describes cell fixation and chromatin preparation from one 15 cm plate (approximately 1.5 x 107 cells). We recommend using 20 ml growth medium per 15 cm plate. Please refer topage 11 for information on scaling the protocol for use with other amounts of cells.
1.

Prepare 15 cm plates for each cell line to be tested. Grow the cells to 70-80% confluency.
Stimulate cells as desired to activate the pathway of interest.

2.

Freshly prepare Complete Cell Fixation Solution for each 15 cm plate. The volumes listed in
the protocol below are enough to process one 15 cm plate. Please refer to the chart on
page 8 to scale the solution volumes.

3.

To fix cells, add 1/10 growth medium volume of freshly prepared Complete Cell Fixative Solution to the existing culture media for the cells (e.g. 20 ml growth medium would get
2 ml Complete Cell Fixation Solution). Shake gently at room temperature for 15 minutes.

4.

Stop the fixation reaction by adding 1/20 media volume of Stop Solution to the existing
culture media for the cells (e.g. 20 ml growth medium would get 1.1 ml Stop Solution). Swirlto mix and incubate at room temperature for 5 minutes.

5.

Following the incubation, hold the plate at an angle and using a rubber policeman scrape
cells down to collect them at the bottom edge of the plate. Use a pipette to transfer thecells to a 50 ml conical tube on ice.

6.

Pellet the cells from step 5 by centrifugation for 3 minutes at 1,250 x g at 4°C.

7.

Remove the supernatant and discard. Resuspend the pellet(s) in 10 ml ice-cold PBS Wash Buffer by pipetting up and down. Keep samples ice-cold for the remainder of the procedure.

8.

Centrifuge for 3 minutes at 1,250 x g at 4°C. Remove the supernatant and discard. Wash thepellet(s) a second time in 10 ml ice-cold PBS Wash Buffer by pipetting up and down. Centrifuge for 3 minutes at 1,250 x g at 4°C. Remove the supernatant and discard. (Cell pellets may
be stored at -80°C at this stage).

9.

Resuspend each pellet(s) in 5 ml Chromatin Prep Buffer supplemented with 5 µl PIC and5 µl 100 mM PMSF. Pipet up and down to mix.

10. Incubate on ice for 10 minutes.
11.

Transfer the resuspended pellets individually to a chilled dounce homogenizer on ice. Use
the tight fitting pestle (Type A) to homogenize the sample for 30 strokes. Transfer the contents to a new 15 ml conical tube and centrifuge for 3 minutes at 1,250 x g at 4°C.

12.

Remove the supernatant and discard. Resuspend each pellet in 500 µl ChIP Buffer supplemented with 5 µl PIC and 5 µl 100 mM PMSF. Transfer the contents to a new 2 ml microcentrifuge tube.

13.

Incubate on ice for 10 minutes. Proceed to Step B: Chromatin Sonication of Cultured Cells.

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15Section B. Chromatin Sonication of Cultured Cells
The section below describes the fragmentation of chromatin using sonication. Sonication results
may vary depending on cell type and sonication device being used. This protocol has beenvalidated using Active Motif’s EpiShear™ Probe Sonicator in combination with an EpiShear™ Cooled
Sonication Platform to maintain probe height and temperature consistency between samples. We
do not recommend sonication of samples containing less than 100,000 cells and/or 350 µl volume.
1.

Place the 2 ml microcentrifuge tube containing the chromatin into the tube cooler or packed
ice. Open cap and submerge the microtip into the liquid until the microtip is approximately
5 mm from the bottom of the tube. Sonicate according to optimized settings for the cell
type being used. A recommended starting range for cultured cells is: 25% amplitude, pulse
for 30 seconds on and 30 seconds off for a total sonication “on” time of 10 minutes (which isequivalent to 20 minutes elapsed time).

2.

Spin tubes at 4°C in a microcentrifuge at maximum speed for 2 minutes to pellet the cellulardebris.

3.

Transfer 25 µl of each chromatin preparation into a 250 µl PCR tube for analysis of shearing
efficiency and chromatin quantification. This sample will be used to generate the Input DNA.

4.

Aliquot the remainder of each chromatin preparation into 1.5 ml microcentrifuge tubes. We
recommend making aliquots of 150 µl volume and storing at -80°C.
Note:

The size of the chromatin sonication should be verified before proceeding to theimmunoprecipitation step.

Input Preparation
5. To each 25 µl chromatin preparation from Section B, Step 3 above, add 175 µl TE pH 8.0 and
1 µl RNAse A. Cap the PCR tubes and vortex to mix
6.

Incubate in a thermocycler at 37°C for 30 minutes.

7.

Add 2 µl Proteinase K to each tube and vortex. Incubate tubes in a thermocycler at 55°C for
30 minutes and then increase the temperature to 80°C for 2 hours.

8.

Transfer each chromatin input to a 1.5 ml microcentrifuge tube. Add 83 µl Precipitation Buffer, 2 µl Carrier and 750 µl absolute ethanol. Vortex to mix and chill at -80°C for 30 minutes
to overnight.

9.

Spin at 4°C in a microcentrifuge at maximum speed for 15 minutes.

10. Carefully remove the supernatant taking care not to disturb the pellet. Wash the pellet with
500 µl 70% ethanol and spin at 4°C in a microcentrifuge at maximum speed for 5 minutes.
11.

Carefully remove the supernatant taking care not to disturb the pellet. Remove residual
ethanol with a pipet tip. Leave the tubes uncapped and air dry for 10-15 minutes.

12.

When the pellets are dry, add 25 µl DNA Purification Elution Buffer to each tube. Incubate atroom temperature for 10 minutes. Then vortex to ensure the pellet is completely resuspended. This solution contains your Input DNA.
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16

13.

Read the absorbance of each sample on a NanoDrop or other spectrophotometer at
260 nm to determine the DNA concentration of each chromatin preparation. Set aside
500 ng of DNA for analysis on an agarose gel. Store the remaining Input DNA at -20°C.

14.

Analyze each chromatin preparation on an agarose gel.

15.

a.

Prepare 500 mM NaCl by adding 2 µl 5M NaCl to 18 µl sterile water. Vortex to mix.

b.

Transfer 500 ng of Input DNA to a 250 µl PCR tube and add 1 µl 500 mM NaCl. Adjust
the final volume to 10 µl with sterile water if needed.

c.

Heat samples in a thermocycler at 100°C for 20 minutes followed by ramping the temperature down to 50°C.

d.

Remove tubes from the thermocycler and incubate at room temperature for 5 minutes.

e.

Add gel loading buffer to each sample and run on a 1.5% agarose gel. Include a 100
bp DNA ladder to analyze chromatin size. DNA should appear as a smear anywhere
between 200-1200 bp.

If chromatin preparations were successful, the aliquots stored at -80°C from Section B, Step 4
can be used to perform the ChIP reactions in Section E.

Figure 1: Validation of chromatin shearing efficiency.
Three chromatin preparations of MCF-7 cells were fixed and sonicated using the EpiShear™ Probe Sonicator and EpiShear™
Cooled Sonication Platform from Active Motif. The chromatin was prepared according to the instructions in the manualand 500 ng each was run on a 1.5% agarose gel. The three samples show a DNA smear concentrated around 200-500 bp.

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17Section C: Cell Fixation Starting with Fresh or Frozen Tissue
This protocol describes cell fixation and chromatin preparation from 100-400 mg fresh or frozen
animal tissue. If performing chromatin preparation on multiple tissue samples, we recommend
completing Steps 1-7 for each sample before processing the next sample.
1.

For tissue fixation, transfer 10 ml Complete Tissue Fixation Solution (see Buffer Preparation
on page 9) to a 60 mm petri dish. Place the dish on ice.

2.

Add 100-400 mg fresh or frozen tissue sample to the petri dish and ensure that the sample isfully immersed. Cut the tissue sample into small pieces (approximately 1 mm cubes) using arazor blade.

3.

Transfer the sample plus the Complete Tissue Fixation Solution to a 15 ml conical tube androtate at room temperature for 15 minutes.

4.

Stop the fixation reaction by adding 515 µl Stop Solution to the conical tube and rotate atroom temperature for 5 minutes.

5.

Place the conical tube on ice and homogenize the contents with a hand-held tissue homogenizer set at 30,000 rpm for 45 seconds.

6.

Pellet the cells from step 5 by centrifugation for 3 minutes at 1,250 x g at 4°C.

7.

Remove the supernatant and discard. Resuspend the pellet in 10 ml ice-cold PBS Wash Buffer
by pipetting up and down. Keep samples ice-cold for the remainder of the procedure.

8.

Centrifuge for 3 minutes at 1,250 x g at 4°C. Remove the supernatant and discard. Wash thepellet(s) a second time in 10 ml ice-cold PBS Wash Buffer by pipetting up and down. Centrifuge for 3 minutes at 1,250 x g at 4°C. Remove the supernatant and discard. (Cell pellets may
be stored at -80°C at this stage).

9.

Resuspend each pellet in 5 ml Chromatin Prep Buffer supplemented with 5 µl PIC and5 µl 100 mM PMSF.

10. Incubate on ice for 10 minutes.
11.

Transfer the resuspended pellet(s) individually to a chilled dounce homogenizer on ice. Use
the tight fitting pestle (Type A) to homogenize the sample for 30 strokes. Once finished,transfer the contents to a new 15 ml conical tube.

12.

Centrifuge for 3 minutes at 1,250 x g at 4°C.

13.

Remove the supernatant and discard. Resuspend each pellet in 500 µl - 1 ml ChIP Buffer
supplemented with PIC and 100 mM PMSF. (For 500 µl add 5 µl PIC and 5 µl PMSF. For 1 ml
add 10 µl PIC and 10 µl PMSF.) Transfer the contents to a new 2 ml microcentrifuge tube.

14.

Incubate on ice for 10 minutes. Proceed to Section D: Chromatin Sonication of Tissue.

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18Section D. Chromatin Sonication of Tissue
The section below describes the fragmentation of chromatin using sonication. Due to theincreased concentration of protein and cellular debris present in animal tissue, we recommend
following this protocol for the preparation of chromatin and input DNA from tissue. Sonication results may vary depending on tissue type and sonication device being used. This protocol has beenvalidated using Active Motifs EpiShear™ Probe Sonicator in combination with the EpiShear™ Cooled
Sonication Platform to maintain probe height and temperature consistency between samples. We
do not recommend sonication of samples containing less than 50 mg tissue and/or 350 µl volume.
1.

Place the 2 ml microcentrifuge tube containing the chromatin into the tube cooler or packed
ice. Open cap and submerge the microtip into the liquid until the microtip is approximately
5 mm from the bottom of the tube. Sonicate according to optimized settings for the tissue
type being used. A recommended starting range for tissue samples is: 25% amplitude, pulse
for 30 seconds on and 30 seconds off for a total sonication “on” time of 10 minutes (which isequivalent to 20 minutes elapsed time).

2.

Spin tubes at 4°C in a microcentrifuge at maximum speed for 2 minutes to pellet the cellulardebris.

3.

Transfer 25 µl of each chromatin preparation into a 250 µl PCR tube for analysis of shearing
efficiency and chromatin quantification. This sample will be used to generate the Input DNA.

4.

Aliquot the remainder of each chromatin preparation into 1.5 ml microcentrifuge tubes. We
recommend making aliquots of 150 µl volume and storing at -80°C.
Note:

The size of the chromatin sonication should be verified before proceeding to theimmunoprecipitation step.

Input Preparation
5. To each 25 µl chromatin preparation from Section D, Step 3 above, add 175 µl TE pH 8.0 and
2 µl RNAse A. Cap the PCR tubes and vortex to mix.
6.

Incubate in a thermocycler at 37°C for 1 hour.

7.

Add 5 µl Proteinase K to each tube, vortex and incubate in a thermocycler at 37°C for
3 hours.

8.

Add 10 µl 5 M NaCl, vortex and incubate at 65°C for 6-16 hours to reverse cross-links.

9.

Remove tubes from the thermocycler and add 250 µl phenol and 125 µl chloroform:isoamyl
alcohol (24:1). Vortex vigorously and spin tubes in a room temperature microcentrifuge at
maximum speed for 2 minutes.

10. Transfer each upper aqueous layer to a new 1.5 ml microcentrifuge tube and add 250 µl
chloroform:isoamyl alcohol (24:1). Vortex vigorously and spin tubes in a room temperature
microcentrifuge at maximum speed for 2 minutes.
11.

Transfer the upper aqueous layer to a new 1.5 ml microcentrifuge tube. Add 83 µl Precipitation Buffer, 2 µl Carrier and 900 µl absolute ethanol. Vortex to mix and chill at -80°C for 30
minutes to overnight.
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19

12.

Spin at 4°C in a microcentrifuge at maximum speed for 15 minutes.

13.

Carefully remove the supernatant taking care not to disturb the pellet. Wash the pellet with
500 µl 70% ethanol and spin at 4°C in a microcentrifuge at maximum speed for 5 minutes.

14.

Carefully remove the supernatant taking care not to disturb the pellet. Remove residual
ethanol with a pipet tip. Leave the tubes uncapped and air dry for 10-15 minutes.

15.

When the pellets are dry, add 25 µl DNA Purification Elution Buffer to each tube. Incubate atroom temperature for 10 minutes. Then vortex to ensure the pellet is completely resuspended. This solution contains your Input DNA.

16.

Read the absorbance of each sample on a NanoDrop or other spectrophotometer at
260 nm to determine the DNA concentration of each chromatin preparation. Set aside
500 ng of DNA for analysis on an agarose gel. Store the remaining Input DNA at -20°C.

17.

Analyze each chromatin preparation on an agarose gel.
a.

Prepare 500 mM NaCl by adding 2 µl 5M NaCl to 18 µl sterile water. Vortex to mix.

b.

Transfer 500 ng of Input DNA to a 250 µl PCR tube and add 1 µl 500 mM NaCl. Adjust
the final volume to between 10 µl with sterile water if needed.

c.

Heat samples in a thermocycler at 100°C for 20 minutes followed by ramping the temperature down to 50°C.

d.

Remove tubes from the thermocycler and incubate at room temperature for 5 minutes.

e.

Add gel loading buffer to each sample and run on a 1.5% agarose gel. Include a 100
bp DNA ladder to analyze chromatin size. DNA should appear as a smear anywhere
between 200-1200 bp. (See Figure 1 on page X as an example)

18. If chromatin preparations were successful, the aliquots stored at -80°C from Section D,
Step 4 can be used to perform the ChIP reactions in Section E.

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20Section E. Immunoprecipitation
Successful chromatin immunoprecipitation depends on the quality of the ChIP antibody and theabundance of the target protein. We suggest using 30 µg chromatin (4.5 x 106 cell equivalents) per
immunoprecipitation reaction to ensure good ChIP DNA recovery and successful library preparations, although 10 µg may be used for highly abundant histone target proteins.
1.

Thaw sonicated chromatin on ice. Spin chromatin at 4°C in a microcentrifuge at maximum
speed for 2 minutes.

2.

Set up the ChIP reactions by adding the components in the order shown in Table 1 below
to 1.5 ml microcentrifuge tubes. Be sure to use the DNA concentration that was determinedfor your sonicated chromatin sample to calculate the volume to use.

3.

In a 1.5 ml microcentrifuge tube prepare the antibodies to be used in the ChIP reactions.
Use a separate tube for each antibody. To the tube add 5 µl Blocker and 4 µg ChIP antibody.
(Antibody volume should not exceed 30 µl per reaction). Incubate Antibody/Blocker mix for
1 minute at room temperature and then add to the ChIP reactions.
Table 1
Reagent

1 reaction

Sheared Chromatin (10 - 30 µg)

X µl

ChIP Buffer

adjust up to 200 µl

Protease Inhibitor Cocktail (PIC)

5 µl

Antibody/Blocker mix (from Step 3 above)

not to exceed 35 µl

Maximum Volume Allowed

240 µl4.

Cap tubes and incubate on an end-to-end rotator overnight at 4°C.

5.

The Protein G agarose beads require washing before use. Transfer 30 µl Protein G agarose
beads for each IP reaction to a 1.5 ml microcentrifuge tube. Add an equal volume of TE,
pH 8.0 and invert to mix. Spin at 1250 x g in a microcentrifuge for 1 minute. Remove thesupernatant equivalent to the volume of TE added to the agarose beads.
Note:

Before pipetting the Protein G agarose beads, they should be fully resuspended
by inverting the tube. When pipetting the beads, cut 2 mm from the end of apipet tip to prevent the tip from becoming clogged.

6.

Wash the beads a second time with the same volume of TE, pH 8.0. Invert to mix. Spin at
1250 x g for 1 minute in a microcentrifuge. Remove the supernatant equivalent to the volume
of TE added to the agarose beads. The beads are now ready to use.

7.

Spin the ChIP reactions at 1250 x g for 1 minute to collect liquid from the inside of the cap.

8.

Using a cut pipet tip, add 30 µl washed Protein G agarose beads to each immunoprecipitationwww.activemotif.com

21

reaction. Cap tubes and incubate on an end-to-end rotator at 4°C for 3 hours.
9.

Label a ChIP Filtration Column for each ChIP reaction. Remove the tab from the bottom of
the column and place in an empty 1 ml pipet tip box as a holder (see Figure 2 below).

10. Remove ChIP reactions from rotator and spin at 1250 x g for 1 minute to collect liquid frominside of the cap.
11.

Add 600 µl ChIP Buffer to each ChIP reaction then transfer the entire reaction (including theprotein G agarose beads) to its labeled column. Allow flow-through to occur by gravity.

12.

During the gravity flow, transfer 100 µl per ChIP reaction of Elution Buffer AM4 to a 1.5 ml
microcentrifuge tube and allow to pre-warm at 37°C during the wash steps.

13.

Wash each column with 900 µl Wash Buffer AM1. Let stand for 3 min.

14.

Repeat Step 13 four more times for a total of five washes.

15.

Transfer columns to a new 1.5 ml microcentrifuge tube and spin in a room temperature
microcentrifuge at 1250 x g for 3 minutes to remove residual Wash Buffer.

16.

Following the spin, transfer the ChIP Filtration Columns to new 1.5 ml microcentrifuge tubes.
Add 50 µl 37°C Elution Buffer AM4 to each column. Incubate at room temperature for5 minutes. Spin in a room temperature microcentrifuge at 1250 x g for 3 minutes.

17.

With columns remaining in the same microcentrifuge tube, add another 50 µl 37°C Elution
Buffer AM4 to each column. Incubate at room temperature for 5 minutes and spin in a room
temperature microcentrifuge at 1250 x g for 3 minutes.

18. Discard the ChIP Filtration Columns. The flow-through (~100 µl volume) contains the ChIP
DNA. Proceed to Section F: Reversal of Cross-links and DNA Purification.

Figure 2: Using the ChIP Filtration Columns.
Remove the tab from the bottom of the ChIP Filtration Columns and place columns in an empty 1 ml pipet tip box toperform the wash steps.

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22Section F. Reversal of Cross-links and DNA Purification
1.

Transfer each eluted ChIP DNA to a 250 µl PCR tube and add 2 µl Proteinase K. Vortex to mix
and heat in a thermocycler at 55°C for 30 minutes and then increase the temperature to 80°C
for 2 hours.

2.

Transfer the DNA to a 1.5 ml microcentrifuge tube and add 5 volumes (500 µl) DNA Purification Binding Buffer to each tube and vortex to mix. Adjust the pH with 5 µl 3M Sodium
Acetate. The sample should be bright yellow in color to indicate a proper pH. If your sample
is not bright yellow, please refer to the Troubleshooting guide in the Appendix on page 31 fordetails to adjust pH prior to loading the sample into the purification column.

3.

For each sample, place a DNA purification column (AM #103928) in the collection tube andadd each pH adjusted sample to its own column. Close the cap on each column, place themwith the collection tubes in a microcentrifuge and spin them at 14,000 rpm for 1 minute.

4.

Remove the column from the collection tube, then remove and discard the flow throughfrom the collection tube. Return the column to the collection tube.

5.

Prepare DNA Purification Wash Buffer (AM #103497) before the first use. Follow the instruction on page 10 for the addition of ethanol prior to using the solution. Add 750 µl DNA Purification Wash Buffer to each column and cap the column.

6.

Spin at 14,000 rpm for 1 minute in a microcentrifuge.

7.

Remove the column from the collection tube, then remove and discard the flow throughfrom the collection tube. Return the column to the collection tube.

8.

With the column cap open, spin at 14,000 rpm for 2 minutes in a microcentrifuge to remove
any residual Wash Buffer from the column.

9.

Transfer the column to a clean microcentrifuge tube. Pre-warm the required elution buffer
volume at 37°C for 5 minutes prior to use.

10. Add 36 µl of 37°C DNA Purification Elution Buffer (AM #103498) to the center of the column
matrix and incubate for 1 minute at room temperature. Spin at 14,000 rpm for 1 minute in a
microcentrifuge.
11.

Discard column. Purified DNA may be stored at -20°C for future use.

Optional: Validation of ChIP-enriched DNA
The quality of the ChIP-enriched DNA can be evaluated using Active Motif’s ChIP-IT™ qPCR
Analysis Kit (Catalog No. 53029). Gene-specific positive and negative control primer sets should
be included in the analysis to determine the fold enrichment. Dilute 6 µl ChIP DNA in 94 µl DNA
Purification Elution Buffer and use 5 µl per qPCR reaction according the instructions provided in
the ChIP-IT qPCR Analysis Kit.

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23

Module 2 – Sequencing Library Preparation
This module is designed to add methylated adapters to the ends of the ChIP-enriched DNA toprepare a sequencing library. These adapters contain methylated cytosine residues throughout
the adapter sequence. This prevents conversion of the cytosines during the subsequent bisulfite
conversion treatment in Module 3, thereby preserving the original adapter sequence for library
amplification. Please refer to pages 9 of the manual for the kit components and additional materials required to complete the sequencing library preparation.

Reagent Information
Quick spin the vials before opening.
Enzymes used for library construction are temperature sensitive and should be kept on ice during
the entire process and returned to -20°C storage as quickly as possible.

Section G: Perform End Repair
This process is to convert the overhangs into phosphorylated blunt ends.
1.

Prepare a fresh 1:5 dilution of the DNA Polymerase I Klenow just before use. Add 0.5 µl DNA
Polymerase I Klenow (5 U/µl) to 2 µl sterile dH2O. Pipet up and down to mix. Discard any
unused enzyme dilution.

2.

Using 200 µl PCR tubes, prepare end repair reactions on the purified ChIP DNA.
Reagents

One rxn

ChIP enriched DNA (from Section F)

30 µl

dH2O

Up to 40 µl

10X T4 DNA Ligase Buffer

5 µl

dNTP Mix, 10 mM each

2 µl

T4 DNA Polymerase (3 U/µl)

1 µl

Diluted DNA Polymerase I Klenow (5 U/µl) from Step K.1

1 µl

T4 Polynucleotide Kinase (10 U/µl)

1 µl

Total Volume

50 µl

3.

Incubate the reactions in a thermal cycler for 30 minutes at 20°C.

4.

Purify the reactions using QIAquick PCR Purification Kit (QIAGEN Cat. No. 28104) following
the suppliers protocol. Elute the DNA using 36 µl Buffer EB.

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24Section H: Add ‘A’ Overhang to 3´ Ends of DNA Fragments
This step is designed to add an ‘A’ base to the 3´ end of the blunt phosphorylated DNA fragments
to prepare the DNA fragments for ligation to methylated adapters.
1.

Prepare reactions to add an ‘A’ overhang to the 3´ ends of the DNA fragments.
Reagents

One rxn

DNA from Step G.4

34 µl

10X Reaction Buffer AM3

5 µl

1 mM dATP

10 µl

Klenow Fragment (3´-5´ exo-) (5 U/µl)

1 µl

Total Volume

50 µl

2.

Incubate the reactions in a thermal cycler for 30 minutes at 37°C.

3.

Purify the reactions using the MinElute PCR Purification Kit (QIAGEN Cat. No. 28004) following the suppliers protocol. Elute the DNA using 11 µl Buffer EB. Perform a second elution using
11 µl Buffer EB for a total elution volume of 23 µl.

Section I: Ligate Methylated Adapters to DNA Fragments
This protocol is for ligation of methylated adapters to the ends of the DNA fragments.
1.

Methylated adapters are provided ready for use. These adapters are compatible with Illumina
sequencing platforms. Please see Appendix Section O for adapter sequence information.

2.

Set up adapter ligation reactions as follows:
Reagents

One rxn

DNA from Step H.3

21 µl

2X Quick Ligation Buffer

25 µl

5 µM Methylated Adapter Mix

3 µl

T4 DNA Ligase (2,000 U/µl)

1 µl

Total Volume

50 µl

3.

Incubate for 20 minutes at room temperature (approximately 24°C).

4.

Purify the reactions using MinElute PCR Purification Kit (QIAGEN Cat. No. 28004) according
to the suppliers protocol. Elute the DNA using 11 µl Buffer EB.

5.

Proceed to Module 3 to perform the bisulfite conversion reaction.

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25

Module 3 – Bisulfite Conversion
In this module, the adapter-ligated ChIP-enriched DNA will undergo bisulfite conversion to determine the methylation status of the enriched DNA at single-base-pair resolution. Please refer topages 10 of the manual for the kit components and additional materials required to complete thebisulfite conversion. This protocol calls for the use of Glycogen as a carrier, this component wasshipped with the -20°C storage items in Module 2.

Reagent Information
Preparation of Conversion Buffer
Two tubes of Conversion Reagent are provided in the Bisulfite Conversion Kit. Each vial is sufficient for performing 10 bisulfite conversions. Prepare the Conversion Buffer by resuspending oneof the Conversion Reagent tubes with 875 µl dH2O, and 350 µl Buffer A. Mix at room temperaturefor 10 minutes with intermittent vortexing.
NOTE:

This is a saturated solution and it is normal for the Conversion Reagent to notdissolve completely. The Conversion Buffer is stable for one week at -20°C.
Conversion Reagent is sensitive to air and moisture. Keep cap on tight and re-cap
immediately after use.

Preparation of Hydroquinone
Hydroquinone is an alkalizing agent and prevents DNA from strand breakage because of
depurination. Each tube provided can be used for 10 conversion reactions. Resuspend one tube
with 100 µl dH2O. Keep mixture in dark and discard after use.
Buffer B
Prepare a 3-fold dilution of Buffer B by adding 10 µl Buffer B to 20 µl dH2O. Store at RT.
Preparation of Desulfonation Buffer
For each reaction, combine 22 µl Buffer B with 88 µl dH2O and 110 µl 100% isopropanol. If thisreagent will not be used immediately, it can be stored for up to one week at -20°C.
DNA Binding Buffer / DNA Wash Buffer / DNA Elution Buffer
Are supplied ready to use.
Glycogen (20 mg/ml)
This item was shipped with the Module 2 components and should be stored at -20°C. Prepare a
1 mg/ml working solution of glycogen by adding 2 µl Glycogen stock to 38 µl dH2O. The diluted
stock may be stored at -20°C for future use.

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26Section J: Conversion Reaction
1.

Prepare Conversion Buffer and Hydroquinone as described in the Buffer Preparation Section
on page 26.

2.

Set up a conversion reaction for each sample by adding the reagents in the order listed below into PCR tubes. Mix well by pipetting. If using less than 10 µl of DNA, adjust the volume
with water to a final volume of 13 µl.
Reagent

Quantity

DNA from Step I.6

10 µl

Diluted Buffer B

3 µl

Total Volume

13 µl

3.

Place tubes in the thermocycler and incubate at 90°C for 2 minutes. Transfer samples immediately to ice.

4.

Add Hydroquinone and Conversion Buffer to the PCR tubes while still on ice to a final
volume of 140 µl. Keep hydroquinone away from light.
Reagent

5.

Denatured DNA

13 µl

Hydroquinone

7 µl

Conversion Buffer

120 µlTotal Volume

140 µlPlace tubes in the thermocycler and set the following program. To prevent evaporation ofsamples, use the heated lid or overlay reactions with mineral oil.

		6.

Quantity

94°C for 3 minutes
50°C for 5 hours
Hold at 4°C

Remove tubes and continue with Step K below. The protocol can also be stopped here andDNA samples can be kept at 4°C in the dark for up to 5 days.

Section K: On-column Desulfonation and DNA Purification1.

Prepare Desulfonation Buffer as described in the Buffer Preparation section on page 26.

2.

For each conversion reaction, aliquot 500 µl DNA Binding Buffer into a 1.5 ml microcentrifuge
tube. Add the entire conversion reaction mixture to the DNA Binding Buffer in the tube. Add
1 µl Glycogen (1 mg/ml) to each tube and vortex to mix.

3.

Place the desired number of DNA purification columns into collection tubes.

4.

Pipet each DNA Binding Buffer/conversion reaction mixture into one of the DNA Purificationwww.activemotif.com

27

columns and spin at 10,000 rpm for 30 seconds in a microcentrifuge. Empty the collection
tube and place the column back on the collection tube.
5.

Add 200 µl DNA Wash Buffer to each column and spin at 10,000 rpm for 30 seconds in a
microcentrifuge.

6.

Add 200 µl Desulfonation Buffer to each column and incubate 20 minutes at room temperature. Spin at 10,000 rpm for 30 seconds in a microcentrifuge. Empty the collection tube andplace the column back on the collection tube.

7.

Add 200 µl DNA Wash Buffer to each column and spin at 10,000 rpm for 30 seconds in a microcentrifuge. Empty the collection tube and place the column back on the collection tube.

8.

To remove any residual wash buffer, spin at 10,000 rpm for 30 seconds in a microcentrifuge.
Remove the column and place in a new 1.5 ml microcentrifuge tube.

9.

Add 24 µl DNA Elution Buffer directly to the filter of the column.

10. Incubate 3 minutes at room temperature. Spin at 10,000 rpm for 30 seconds in a microcentrifuge. The eluate will contain the converted DNA.
11.

The eluted DNA is now ready for PCR amplification.

Section L: Enrich the Adapter-Modified DNA Fragments by PCRThis step is designed to PCR amplify the bisulfite converted DNA. We recommend using a DNA
polymerase that is designed to amplify bisulfite converted DNA (e.g. KAPA HiFi HotStart Uracil+
Ready Mix, KAPA Biosystems Catalog No. KK2801). PCR primers should be compatible with Illumina
sequencing platforms. Primers for single, paired-end or multiplex sequencing may be used. For
information on primer sequences, please refer to Appendix Section O.
1.

2.

Using a 200 µl PCR tube, prepare the following reactions. Add components in the order listed
in the table below.
Reagents

One rxn

Adapter Ligated DNA

23 µl

PCR primer mix (10 µM each)

2 µl

2X High Fidelity Uracil+ Mix

25 µl

Total Volume

50 µl

Amplify the reactions in a thermal cycler according to the recommendations for the DNApolymerase being used. For example with the KAPA HiFi HotStart Uracil+:
98°C for 45 seconds(98°C for 15 seconds, 65°C for 30 seconds and 72°C for 30 seconds) for 14 cycles
72°C for 1 minuteHold at 4°C
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28Section M: Size Select the LibraryThis process removes excess adapters and selects a size range of DNA templates.
1.

Prepare a large comb 1.5.% agarose TAE gel with 400 ng/ml ethidium bromide or equivalent.
Note:

Ethidium bromide is a mutagen. Use appropriate precaution when handling.

2.

Add 2.5 µl loading buffer to each reaction.

3.

Run the entire adapter ligated DNA from Step L.2 and 500 ng of 100 bp DNA ladder on a 1.5%
agarose gel for size selection of the DNA library. To avoid potential cross contamination of
adjacent wells, leave at least one empty lane between DNA ladder, sample and Input wells.

4.

Run the gel at 120V until the tracking dye is 2/3 down the length of the gel.

5.

Visualize the DNA using a Dark Reader transilluminator to avoid exposure to UV light. If
using a UV light, work quickly when excising the band to minimize direct exposure to UV.
Prolonged exposure to UV light can damage DNA.

6.

Using a clean razor blade, excise a gel slice in the 200-300 bp range. We recommend tophotograph the gel before and after the band is excised.

7.

Use a QIAquick Gel Extraction Kit (QIAGEN Cat. No. 28704) to purify the DNA from the agarose. Make the following protocol modifications to the QIAquick Gel Extraction Kit protocol:

8.

a.

QIAquick columns can handle a maximum of 400 mg agarose. If using a larger quantityof agarose, you will need to split your sample across multiple columns.

b.

Incubate the gel slice in 3 volumes Buffer QG at 37°C for 30 minutes instead of thesuggested 50°C for 10 minutes. Buffer volumes and incubation times are dependent
upon the thickness of the gel and may need to be adjusted based on your sample. If
necessary, add additional Buffer QG and increase the duration of the 37°C incubation.

c.

Add the recommended extra 0.5 ml Buffer QG to the QIAquick column.

d.

During the wash steps, incubate 2-5 minutes in Buffer PE before centrifugation.

e.

Elute the DNA in 38 µl Buffer EB.

Determine the final concentration of the library using Picogreen or equivalent fluorescentquantitation method. Libraries are now ready for sequencing.

Section N: Analyzing the Sequencing Results
Analysis of bisulfite converted DNA can be difficult given the lack of complexity of the DNA bases
following conversion. As a result, shorter read length may be more difficult to accurately map. It
is advised to generate longer reads and use a software program designed for analysis of genomewide bisulfite converted DNA. Several programs exist for bisulfite-sequencing analysis, such as
the freeware program GBSA (see reference http://www.ncbi.nlm.nih.gov/pubmed/23268441), or
Bis-SNP (see reference http://epigenome.usc.edu/publicationdata/bissnp2011/).
1.

If the CpG dinucleotide was methylated in the original DNA, the sequencing read will show
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29

a CG at that location. If the CpG dinucleotide was unmethylated in the original DNA, thebisulfite conversion will have converted the cytosine to uracil and the sequencing read willshow a TG at that location.
2.

Compare the bisulfite converted sequence data against the unconverted DNA sequence todetermine the percentage of methylation.

Appendix
Section O. Adapter and Primer Selection
The ChIP-Bis-Seq Kit includes methylated adpaters for use in generating Next Generation Sequencing libraries. Methylated adpaters are provided as a 5 µM mix and should be used according
to the recommendations in the manual. Information regarding the sequence of the methylated
adapters and corresponding primer sequences for use in the PCR amplification of the adapterligated DNA can be found on the Illumina website at http://support.illumina.com/downloads/
illumina-customer-sequence-letter.html. Download the customer sequence letter and go to thesection for Oligonucleotide sequences for Genomic DNA.
Since methylated adapters were used, the Oligonucleotide sequence for Genomic DNA adaptersis retained following bisulfite conversion. Select a corresponding PCR primer sequence for use insingle, paired-end or multiplex analysis to fit your sequencing needs. Due to the lack of complexity with bisulfite converted DNA, longer sequencing reads (>75 bp) will improve the ability to map
the sequence back to the reference genome.

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Appendix
Section P. Troubleshooting Guide
Problem/question

Recommendation

At what points in the
protocol can I stop the
ChIP protocol?

The protocol may be stopped and samples stored at the times and temperatures below:
1. After formaldehyde fixation and centrifugation (intact cell pellet), -80°C.
2. After chromatin shearing, -80°C.
3. After DNA clean up, -20°C.

After sonication shearing
and centrifugation, aviscous or cloudy layer isvisible in the chromatin.

Depending upon the cell type, lipid or glycogen layers may be seen after centrifugation.
For example, fatty tissues may have a lipid layer. Avoid such layers when you remove thesupernatant. However, if the whole supernatant is cloudy, it should not interfere with the
IP reaction.

Poor yield of shearedchromatin.

Insufficient cell numbers were used. Repeat chromatin preparation using a larger numberof cells.
Nuclei not released. It is highly recommended to perform dounce homogenization, evenwhen using sonication. Use a dounce homogenizer with a small clearance pestle (Active
Motif Catalog Nos. 40401 & 40415). Monitor cell lysis under a microscope. Generally, themore cells that are lysed, the higher the sheared chromatin yield.
Sonicated samples were emulsified. Avoid emulsification by turning up the power ofthe sonicator gradually. If a chromatin preparation becomes emulsified inadvertently,
discontinue shearing and centrifuge the sample for 4 minutes at 8,000 rpm in a 4°C
microcentrifuge to remove trapped air.
Use fresh formaldehyde when preparing Complete Cell Fixation Solution and Complete
Tissue Fixation Solution.
Buffers were not scaled proportionally to the size of the sample. Use the chart in Cell
Growth Recommendations to scale up or down chromatin preparation.

Shearing efficiency is notclear from gel analysis.

Material is stuck in the wells, and smears or streaks are seen from the top to bottom of
the lane. The sheared chromatin needs to have the cross-links reversed, protein removed(Proteinase K) and RNA removed (RNase), followed by DNA purification.
High molecular weight products. Decrease the size of the fragments by re-sonicating thesample.

Performing ChIP with alarge volume of chromatin.

This is not recommended. It is better to set up several small ChIP reactions (240 μl each)and pool the samples at the end, rather than trying to ChIP a single large sample. Do not
perform a single scaled-up reaction, as the capture efficiency is lower.

ChIP DNA does not turnbright yellow following theaddition of 3 M sodium
acetateIf the color is light orange or violet, this indicates the pH is too high. Add more 3 M
sodium acetate 5 µl at a time, mixing after addition until the color is bright yellow. This
step is crucial to the success of DNA binding and purification. For a full color image please
see the manual for Active Motif’s Chromatin IP DNA Purification Kit Catalog No. 58002
available online at our website www.activemotif.com.

High background.

Chromatin not sheared enough. Shearing should produce DNA fragments that are smallenough to exclude background from neighboring chromosomal sequences, but still large
enough that there is a good possibility your amplicon remains intact. We recommend
200-1200 bp fragments. If the DNA fragments are too large, the background is increased.
Consider increasing the number of pulses for sonication. Check the fragment size on a gel
to assess your shearing efficiency.
Antibody issue. Too much antibody relative to the amount of chromatin in the ChIP
reaction. Excess antibody will result in more non-specific binding, which will be detected
as increased background.

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31

Problem/question

Recommendation

Poor or no enrichmentwith target antibody.

Too little chromatin. Generally, we recommend using 30 µg of chromatin per ChIP
reaction for use downstream in bisulfite conversion. ChIP reactions should not exceed
50 µg per IP reaction. Be sure to quantitate the concentration of the sheared chromatin
sample(s) being ChIP’d to ensure both that adequate chromatin is used per sample, andthat equal mass quantities of chromatin are used in each ChIP.
Antibody is not ChIP validated. The antibody does not efficiently recognize fixed
proteins, either because the epitope is destroyed by fixation or because the epitope ismasked by other proteins in a larger complex. To assist in ChIP validating an antibody, it
is very useful to use a positive control antibody such as Histone H3K4me3 (Catalog No.
39915) and a negative IgG from the same species, and primers that have been proven towork in the type of PCR being used. Active Motif offers species-specific ChIP-IT Control
qPCR Kits for antibody validation (Catalog Nos. 53026, 53027 & 53028).
Low-affinity antibody. Use a different antibody.
Antibody affinity to protein G is weak. Individual monoclonals have variable binding affinities to protein G, which are pH dependent; the optimal pH may vary for each IgG, For
those with low to medium affinity, capture efficiency by protein G can be dramatically
improved through use of our Bridging Antibody (Catalog No. 53017). This antibody is arabbit anti-mouse pAb that recognizes all subclasses of mouse immunoglobulins. If yourIgG has a weak/medium affinity to protein A or G, the Bridging Antibody will increase
antibody capture by the beads without increasing background.
Problems with PCR. Confirm the amplified sequence for the positive control primer set isbound by the antibody target. Identify other binding sites.

No PCR products for the
ChIP’d samples (but the
Input DNA yields thecorrect PCR product)

Increase the amount of chromatin used in the ChIP reaction, the amount of antibody
used, or both.

No ChIP PCR productswith real-time PCR

Confirm the species specificity and efficiency of your primers. You may need to redesign
your primers. Primers that work in end point PCR do not always work in qPCR.

Use a different antibody.

No ethanol in DNA Purification Wash Buffer. Make sure that ethanol has been added to
the DNA Purification Wash Buffer prior to first use.
Conversion time

For DNA sequences that are GC-rich and/or contain extensive secondary structures wesuggest a 9 hour conversion at 50°C. Longer conversion times, up to 16 hours, may be
used if noticing incomplete conversion, however, this may result in degradation of theDNA. If noticing a loss of DNA after the conversion reaction, the conversion time can be
reduced to 3 hours at 50°C

Poor PCR amplification

We recommend performing a Hot Start PCR reaction using a polymerase that has beenvalidated to work with bisulfite converted DNA. Follow the recommendations of the
manufacturer for the preparation of the PCR reaction.

Poor sample recovery

When converting a small amount of DNA, add 1 µg Glycogen (20 ng/µl) or tRNA (10 µg/µl) as a carrier to the 500 µl DNA Binding Buffer for each reaction during the on-column
desulfonation and DNA purification step to minimize sample loss.
Quantification of the sequencing library should be performed following PCR amplification. We recommend using a fluorescent quantitation method, such as Picogreen,,todetermine the DNA quantity since the yield recovered may be below the detectable
range of a Nanodrop instrument.

Incomplete conversion

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Conversion reagent is sensitive to air and moisture. If conversion reagent was notproperly sealed and stored, repeat conversion reaction using a new tube of conversion
reagent.

32

Problem/question

Recommendation

Can I use columns to purify my enriched adaptermodified DNA rather than
AMPure® XP beads?

We recommend the use of Agencourt® AMPure® XP Beads (Beckman Coulter Cat No.
A63880) for purification of the adapter-modified DNA rather than column purification.
With column purification we noticed a higher degree of free adapter in the flow through.
The AMPure bead purification provides better purity of the library, but optimization may
be required to determine the optimal amount of beads to use per reaction.

Section Q. Related Products
DNA Methylation		Bisulfite Conversion Kit		
MethylCollector™ Ultra		
HypoMethylCollector™		
MeDIP		Hydroxymethyl Collector™		
hMeDIP		
PvuRts1I restriction enzyme		
b-Glucosyltransferase enzyme		
Recombinant TET1 protein, active		
DNMT Activity / Inhibition Assay		
Methylated DNA Standard Kit		
5-Carboxylcytosine DNA Standard		
Fully Methylated Jurkat DNA		
Jurkat genomic DNA		

Format50 rxns
30 rxns
30 rxns
10 rxns
25 rxns
10 rxns
50 units
500 units
25 µg
1 x 96 rxns
3 x 2.5 µg
0.5 µg
10 µg
10 µg

Catalog No.
55016
55005
55004
55009
55013
55010
55011
55012
31363
55006
55008
55014
55003
55007

Antibodies

FormatCatalog No.

100 µg100 µl100 µl100 µg100 µl50 µg
100 µg200 µl100 µg100 µg100 µg100 µg100 µl200 µg100 µg200 µl100 µg100 µg100 µg100 µg100 µl100 µg100 µl100 µg200 µ1

61111
61225
61223
39999
39769
39649
61255
39203
39204
39205
39206
39207
39907
39365
39215
39547
39216
39217
39218
61291
61443
61389
61395
39741
39625

Application

3-Methylcytosine rabbit pAb
DB
5-Carboxylcytosine rabbit pAb
DB, IF
5-Formylcytosine rabbit pAb
DB, IF
5-Hydroxymethylcytidine mouse mAb
DB, MeDIP
5-Hydroxymethylcytidine rabbit pAb DB, IF, IHC, MeDIP
5-Methylcytosine mouse mAb
DB, FACS, IHC, IP, MeDIP
5-Methylcytosine rabbit pAb
DB, IP, MeDIPCGBP rabbit pAb
WB
DNMT1 mouse mAb
ChIP, IHC, IP, WB
DNMT2 rabbit pAb
WB
DNMT3A mouse mAb
ChIP, IF, IHC, WB
DNMT3B mouse mAb
ChIP, IF, IP, WB
DNMT3L rabbit pAb
WB
Kaiso mouse mAb
WB
MBD1 mouse mAb
WB
MBD2 rabbit pAb
WB
MBD3 mouse mAb
WB
MBD4 rabbit pAb
WB
MeCP2 rabbit pAb
WB
MeCP2 mouse mAb
ChIP, IF, IHC, IP, WB
Tet1 rabbit pAb
ChIP, WB
Tet2 mouse mAb
IP, WB
Tet3 rabbit pAb
WB
Ubiquitin mouse mAb
IF, IHC, IP, WB
Uhrf1 rabbit pAb
WB

For more information, please visit our website at www.activemotif.com.
www.activemotif.com

33

Technical Services
If you need assistance at any time, please call Active Motif Technical Service at one of thenumbers listed below.
Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008
USA
Toll Free:877 222 9543
Telephone: 760 431 1263
Fax:
760 431 1351
E-mail:tech_service@activemotif.com
Active Motif Europe
Avenue Reine Astrid, 92
B-1310 La Hulpe, Belgium
UK Free Phone: 0800 169 31 47
France Free Phone:
0800 90 99 79
Germany Free Phone: 0800 181 99 10
Telephone:		+32 (0)2 653 0001
Fax:		
+32 (0)2 653 0050
E-mail: 		
eurotech@activemotif.com
Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Telephone: +81 3 5225 3638
Fax:
+81 3 5261 8733
E-mail:japantech@activemotif.com
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