THE NEWSLETTER OF ACTIVE MOTIF motif vations APRIL 2016 | VOLUME 17 NUMBER 1

CANCER EPIGENETICS EDITION

Enabling Epigenetics Research Antibodies, Kits and Services for Epigenetics & Gene Regulation Research

Epigenetics & Cancer

Epigenetics, or the heritable changes in gene function that occur independent of DNA sequence, has become a major focus of development and disease research. Countless studies link epigenetic alterations to developmental processes, including X-chromosome inactivation, genomic imprinting, axis patterning and differentiation, as well as to diseases such as cancer, chromosomal instabilities and mental retardation. Because genetics alone does not provide an adequate explanation for the complexity of these processes, a deeper understanding of the role of epigenetics is crucial to unraveling the underlying mechanisms responsible for regulating IN THIS ISSUE development and disease.

2 Epigenetics & Cancer. DNA methylation & gene regulation Methylation & the cancer epigenome 4 RNA Analysis Techniques to Advance DNA methylation is an important Changes in DNA methylation have our Understanding of the Transcriptome regulator of gene expression and been well characterized in cancer. 5 NEW: Tools to Study Nrf2 Transcription chromosome conformation. It occurs In general, the cancer epigenome is Factor Activation at the cytosine bases of eukaryotic marked by global DNA hypomethylation DNA, which are converted to 5-methyl- and promoter-specific DNA hyper- 6 Practical Guide to Chromatin cytosine (5-mC) by DNA methyltrans- methylation, which often leads to Immunoprecipitation Techniques ferase enzymes. DNA methylation silencing of tumor suppressor genes. 10 NEW: Determine CRISPR/Cas9 occurs almost exclusively in the context This link to cancer is supported by the Specificity Using enChIP of CpG dinucleotides that are relatively finding that mutations in DNMT3A are rare in the mammalian genome and found in acute myeloid leukemia, and 11 Quantify Global Changes in 5-mC tend to be clustered in CpG islands. there are currently two FDA approved & 5-hmC in Normal and Diseased Approximately 60% of gene promoters cancer drugs that target DNMTs. The Samples are associated with CpG islands and are recently characterized DNA modifica- 12 A Genome-wide 3’UTR Collection for normally unmethylated. CpG methylation tion 5-hydroxymethylcytosine (5-hmC) High-throughput Functional Screening of gene promoters is usually associated has also been linked to cancer. Both a of miRNA Targets with transcriptional silencing, which reduction in 5-hmC and in expression of can occur through a number of the TET enzymes that convert 5-mC to 13 Validated Antibodies Reveal Histone- mechanisms, including the recruit- 5-hmC have been reported in breast, mediated Effects on miRNA in ment of methyl binding domain (MBD) liver, lung, prostate and pancreatic Non-small Cell Lung Cancer proteins. DNA methylation is involved cancer. 14 High Quality Bromodomain Proteins in a number of cellular functions, such for Developing Robust Assays for as embryonic development, genetic Tools for DNA methylation analysis Epigenetic Drug Discovery imprinting, X chromosome inactivation Active Motif offers a number of products and control of gene expression. Alter- specific for DNA methylation research, ations in normal DNA methylation including kits and antibodies to enrich patterns, either local upregulation for DNA fragments that contain 5-mC at promoters resulting in silencing and 5-hmC. Our DNA methylation- of specific cell cycle regulatory or related antibodies have been validated tumor suppressor genes, or a global for various applications, including decrease in DNA methylation, are methylated DNA immunoprecipitation hallmarks of various types of cancer. (MeDIP) and immunofluorescence.

2 www.activemotif.com EPIGENETICS & CANCER

For complete details on all of our DNA and the first miRNA mimic entered the the functional relevance of a specific methylation products, or to download a clinic for cancer therapy in 2013. miRNA, including the LightSwitch™ free copy of our new DNA Methylation Luciferase Reporter Assay System. & Cancer eBook, please visit us at Tools for miRNA analysis 3’UTR reporter assays have become www.activemotif.com/dnamt. Inherent to the rapid advancements an important component of thorough and general excitement surrounding miRNA target studies because they The role of non-coding RNAs in cancer miRNA discoveries is the need for provide functional evidence for, and The discovery of non-coding RNAs applicable and validated experimental quantitate the effects of, specific (ncRNAs) and their role in gene regu- tools to enable researchers to accurately miRNA-3’UTR interactions in a cell- lation represents a significant advance study the expression and biological based system. For complete details on in cancer research. These ncRNAs function of miRNAs. There are a number all our miRNA products, or to download interact with DNA, RNA or protein of diverse methodologies available for a free copy of our new microRNAs in molecules and/or some combination miRNA detection and target identification, Cancer eBook, please visit us at www. thereof, acting as essential regulators as well as to determine the functional activemotif.com/ncRNAs-and-cancer. in chromatin organization, and tran- effects of a specific miRNA of interest. scriptional and post-transcriptional These include computational tools, Let us do the work for you - regulation. The functional relevance expression and proteomics assays, Active Motif Epigenetic Services of ncRNAs is particularly evident for 3’UTR reporter assays and chromatin Our Epigenetic Services team provides microRNAs (miRNAs) and long non- immunoprecipitation (ChIP) based a wide variety of DNA Methylation and coding RNAs (lncRNAs), which have techniques. Active Motif offers a wide Gene Regulation services. For more been associated with all stages of selection of tools to enable researchers complete information, or to get a quote, cancer including diagnosis, staging, to identify mRNA targets and elucidate visit www.activemotif.com/services. progression, prognosis and response to treatment. Their misexpression 2. Loss of normal repressive Transcription Factors epigenetic marks onco-miRNAs confers upon a cancer cell the capacity miRNA gene 1. CpG promotor for tumor initiation, growth and Genomic loss methylation onco-suppressor Loss of histone acetylation metastasis, making ncRNAs attractive DNA miRNAs therapeutic targets and potentially Aberrant DNA hypermethylation useful diagnostic tools.

MicroRNAs in cancer MicroRNAs are important epigenetic regulators of gene expression and 5 Cap AAA have been shown to play a role in Pri-mRNA numerous biological processes, such Cropping 3. Drosha downregulation as cellular signaling, cell differentiation, growth, development and apoptosis. Mutations and improper regulation of Nucleus Pre-miRNA miRNAs have been linked to a variety of physiological disorders, such as 4. XPO5 mutation XPO5 cancer and heart disease (Figure 1). Cytoplasm The recent emergence of miRNAs has Oncogene been one of the defining developments accumulation 5. DICER1 downregulation in cancer biology. Many studies have 5. TARBP2 mutation demonstrated how miRNAs impact cancer progression through controlling 7. tumor 6. oncogenic suppressor expression of their target mRNAs to miRNA miRNA loss accumulation facilitate tumor growth, invasion, Unwinding of miRNA duplex angiogenesis and immune response. The explosion of knowledge since their Loss of tumor discovery in 1993 has brought forward suppressor genes new diagnostic and therapeutic oppor- tunities. Clinical trials utilizing miRNA Figure 1: DNA methylation and miRNA mysregulation in cancer. profiling for patient prognosis and A schematic representation of alterations in DNA methylation and in the miRNA biogenesis pathway that commonly lead to cancer development clinical response are now underway, (adapted from Bertoli G. et al. (2015). Theranostics 5, 1122–43).

[email protected] 3 TOOLS FOR RNA ANALYSIS

RNA Analysis Techniques to Advance our Understanding of the Transcriptome Technological advances in the field of RNA analysis have expanded our understanding of the transcriptome beyond messenger RNA (mRNA) to introduce new classes of RNAs that include short and long non-coding RNA (ncRNA). These non-coding RNAs vary in cellular localization and appear to have unique regulatory functions. Combining RNA analysis with high quality RNA isolation from subcellular fractions, such as with Active Motif’s RNA Subcellular Isolation Kit, or with next-generation sequencing (NGS) by utilizing Active Motif’s RNA Sequencing (RNA-Seq) Service, enables you to achieve a greater understanding of RNA function in your biological system.

High quality RNA fractions improve data interpretation by reducing the RNA Subcellular Isolation Kit downstream RNA analysis sensitivity of detection for low abundance Active Motif’s RNA Subcellular Isolation transcripts and the ability to study • Separates nuclear & Kit is designed to efficiently isolate sepa- RNA processing dynamics. The RNA cytoplasmic RNA without rate nuclear and cytoplasmic fractions Subcellular Isolation Kit reduces this cross-contamination of RNA for downstream analysis. This complexity to improve downstream method can be used to isolate both long analysis and also provides information • Works with cell or tissue samples and short RNA sequences from cells regarding RNA localization. • Spin columns included for or tissue without cross-contamination RNA purification or the use of phenolic compounds. What’s in the box? Each kit contains enough reagents to • Purified RNA validated for use Traditional methods fall short for RNA perform 15 cytoplasmic and 15 nuclear in RT-qPCR and RNA-Seq analysis because they often isolate total RNA isolations. For more information, RNA which consists of a mixture of visit www.activemotif.com/rna-iso. intronic RNA originating from immature transcripts in the nucleus, mature RNA Product Format Catalog No. from the cytoplasm and a variety of RNA Subcellular Isolation Kit 30 rxns 25501 non-coding RNAs. Such heterogeneity can bias downstream analysis and DNase I Treatment Kit 30 rxns 25502

RNA-Seq Service – get the most out obtain, among other things, information Is polyA selection or rRNA depletion of your RNA-Seq experiment about splice variants, gene fusions appropriate for your experiment? Active Motif provides a comprehensive and SNPs. It is, therefore, important to Active Motif understands and offers RNA-Seq Service that includes design RNA-Seq experiments according solutions for all the experimental everything from isolation of RNA to to the goals in mind. Should you use variables. We will consult with you and data analysis. Combine our worldwide paired-end or single-end sequencing? provide specific recommendations on leading ChIP-Seq Service with RNA-Seq What length of sequencing read is experimental design that precisely to gain an even greater understanding needed, and how deep should you go? align with the goals of your research. of your biological system. RNA-Seq Service Features RNA-Seq is the most widely used technique for studying gene expression • PolyA selection or rRNA reduction • QC performed using Bioanalyzer changes in response to treatment and • Directional library preparation • Data analysis options (TOPHAT, disease, but it also provides much CUFFLINKS, CUFFCOMPARE, more. With sequence data from an • 50bp or 100bp paired-end reads CUFFDIFF) RNA-Seq experiment it is possible to

4 www.activemotif.com TRANSCRIPTION FACTOR SCREENING

NEW Tools to Study Nrf2 Transcription Factor Activation

Nrf2 is an important transcription factor (TF) in the defense against oxidative stress. Much investment has been made in developing Nrf2 agonists for the treatment of diseases such as multiple sclerosis, diabetes and cancer. Like most TFs, Nrf2 activation is dynamically regulated, involving controlled protein degradation and nuclear translocation. Active Motif offers a range of products for Nrf2 research, including TransAM®, a simple, plate-based assay to screen for Nrf2 activation, a chromatin immunoprecipitation (ChIP) validated Nrf2 antibody and compatible ChIP kit, and a luciferase reporter assay system along with pre-cloned Nrf2-related reporter vectors.

1 Figure 1: Nrf2 activation is detected binding. To facilitate your ChIP-Seq Unstimulated using the TransAM Nrf2 Kit. studies of Nrf2, Active Motif provides nm) D,L Sulforaphane Stimulated Active Motif’s Nuclear Extract Kit was a Nrf2 ChIP-Seq validated antibody 450 0.75 used to prepare nuclear lysates from and our best-selling ChIP-IT® High unstimulated HepG2 cells and cells that Sensitivity Kit (Figure 2). Or let Active 0.5 were stimulated for 24 hours with 10 μM D,L-Sulforaphane. The extracts were Motif Epigenetic Services do the exper- ™ assayed using a range of 0.625 to 10 μg iment for you. As the ChIP Experts , 0.25 per well to show the linearity of the with over 10,000 samples processed assay. Robust Nrf2 activation is clearly and the highest level of expertise of Nrf2 activation (OD Nrf2 activation 0 detectable across all conditions. any service provider, you can expect 0.63 1.25 2.5 5 10 the best results. For more information Extract/well (µg) about our custom ChIP-Seq services, visit www.activemotif.com/services. Screening multiple samples? LightSwitch™ Luciferase Reporter Active Motif’s TransAM® Nrf2 Kit includes Assay System that combine optimized a 96-stripwell plate to which an oligomer transfection and luciferase assay corresponding to the antioxidant reagents along with a large selection of response element (ARE) has been Nrf2 responsive luciferase reporter immobilized. Nuclear extracts, prepared constructs. These include a variety of using Active Motif’s Nuclear Extract Kit, synthetic ARE elements and pre-cloned are added to each well. Activated Nrf2 reporter vectors containing the binds the ARE consensus sequence, promoters of Nrf2 responsive genes while unbound proteins are washed such as NQO1 and HMOX1. away. Detection is achieved using an NQO1 HMOX1

Nrf2 primary antibody and HRP- Interested in monitoring Nrf2 Figure 2: ChIP-Seq was performed using Active Motif’s conjugated secondary antibody along activation across the genome? Nrf2 antibody and ChIP-IT High Sensitivity Kit. with colorimetric detection reagents. ChIP-Seq is now the most widely used Select peaks are presented and show Nrf2 binding in the The assay signal is easily measured by technique to study transcription factor promoters of known Nrf2 responsive genes. OD 450nm using a microplate reader (Figure 1). In addition to Nrf2, TransAM Product Format Catalog No. is available for over 40 transcription ® factor targets. For more information TransAM Nrf2 Kit 1 x 96 rxns 50296 and a complete list of TransAM products, Nuclear Extract Kit 100 rxns 40010 go to www.activemotif.com/transam. ARE synthRE 5 μg 32108 NRF2 (NFE2L2) antibody (pAb) 100 μl 61599 Active Motif also offers cell-based screening assays using our ChIP-IT® High Sensitivity Kit 16 rxns 53040

[email protected] 5 CHROMATIN IMMUNOPRECIPITATION

Practical Guide to Chromatin Immunoprecipitation Techniques

Chromatin immunoprecipitation (ChIP) is the most widely utilized technique for analysis of protein-DNA interactions and histone modifications. Given the importance of ChIP and ChIP-Seq data sets for development and disease research, obtaining high quality data is crucial. However, ChIP can be technically challenging, and researchers who are not experts in ChIP techniques are best served using well-validated and reliable kits to perform these assays. As the ChIP experts®, Active Motif has developed a number of highly innovative kits and reagents tailored to aid researchers with their ChIP experiments and to overcome many of the common TOPICS challenges researchers encounter when applying this technique.

Performing ChIP on epigenetic Performing ChIP on epigenetic the ChIP procedure to be completed modifications modifications in a single day. The reduced hands-on As a leader in providing innovative time and use of ChIP-grade magnetic What if I don’t have a sonicator? solutions for ChIP, Active Motif has particles minimize DNA loss and What if I have limited sample material? developed a highly comprehensive sample variation, enabling ChIP to be portfolio of ChIP products and services performed on as few as 100,000 cells. Analyzing low abundance targets to make ChIP technologies accessible With over 500 citations in peer reviewed (transcription factors) to researchers at any level of expertise, publications, this is the method of Choosing the correct antibody and to also provide cutting-edge choice for routine ChIP (Figure 1). technologies to address many of the What if I don’t have a good antibody? common limitations experienced by What if I don’t have a sonicator? Increasing DNA binding site resolution ChIP users when analyzing chromatin The kit comes in two formats, the origi- function. nal ChIP-IT Express sonication protocol How do I normalize for sample bias? and ChIP-IT® Express Enzymatic, the first What interactions are occurring at my Traditional ChIP and ChIP-Seq are ex- kit that enables shearing of chromatin promoter of interest? tremely informative methods for iden- by enzymatic digestion rather than tification and profiling of chromatin sonication for users who do not routinely How can ChIP aid in genome editing? interactions with ubiquitous features, perform ChIP and either do not own a Can I also interrogate the DNA for example chromatin modifying sonicator or are not proficient in its use. methylation status of my ChIP DNA? proteins (e.g. p300, HDACs), histone modifications (e.g. H3K4me3, H3K27me3, To learn more about ChIP-IT Express, Difficult-to-ChIP clinical samples H3K27ac) and transcriptional machinery visit www.activemotif.com/chipit. (e.g. RNA pol II). When conducting Analyzing chromatin-associated RNA ChIP analysis, the most important What if I have limited starting material? NEW: Determine CRISPR/Cas9 aspects of your ChIP protocol are that it Standard ChIP-Seq protocols require specificity using enChIP be reproducible, sensitive and efficient. at least 10 million cells to perform ChIP-Seq, a number that is unattainable For performing ChIP on relatively for many cell types. As a result, one abundant proteins such as histones, of the major obstacles preventing Active Motif’s ChIP-IT® Express kits researchers from generating quality provide the ideal solution. ChIP-IT ChIP-Seq data is the limited cell num- Express kits use magnetic beads to bers available for their experiments. streamline the ChIP protocol, allowing Active Motif’s Epigenetic Services group

6 www.activemotif.com CHROMATIN IMMUNOPRECIPITATION

400 DNA interactions. ChIP filtration gravity What if I don’t have a good antibody?

350 flow columns are used for faster, ChIP analysis is often hindered by the

300 simpler and more consistent capture lack of available antibodies capable of and wash steps. The result is a kit that recognizing fixed protein targets, or 250 is capable of delivering both higher the inability of antibodies to distinguish 200 signals and reduced background levels between protein isoforms. Active Motif 150 when compared to other commercially has developed the Tag-ChIP-IT® Kit to # of Publications 100 available ChIP kits. To learn more enable ChIP without the use of a target- 50 about ChIP-IT High Sensitivity, visit specific antibody. Tag-ChIP-IT utilizes 0

® www.activemotif.com/chipiths. a unique AM-tag that, unlike traditional

Sigma FLAG, GFP or HA tags, is specifically Express Millipore Imprint LowCell# Invitrogen MAGnify™ ® Diagenode Choosing the correct antibody optimized for ChIP. Furthermore, we have T Active Motif Active

Magna ChIP™ Another challenge for epigenetics designed a high specificity antibody to ChIP-I research is the lack of antibodies that the tag to optimize pull-down efficiency Figure 1: ChIP-IT Express is the most highly published have been validated for use in ChIP and during immunoprecipitation (Figure 2). commercial ChIP kit on the market. Comparison of ChIP-Seq. The problem is compounded ChIP-IT Express citations vs. competitor kits. by antibody suppliers who do not The AM-tag is engineered to have mini- manufacture or test the antibodies mal cross-reactivity with mammalian has made significant improvements to they sell, and who sell them to one samples. Additionally, in contrast to our ChIP-Seq protocols and pipeline another and then to researchers. At larger GFP and FLAG tags, the smaller and, as a result, reduced our starting Active Motif, we manufacture and footprint of the AM-tag reduces obstruc- material requirements by 1,000 fold. rigorously test our antibodies in-house tion of the DNA binding domain. The Our Low Cell Number ChIP-Seq Service to ensure their quality and performance. AM-tag design also maximizes exposure enables generation of genome-wide Our ChIP antibodies are validated during the IP reaction to increase the binding profiles from as few as 10K cells. according to the guidelines of the enrichment efficiency of low abundance ENCODE* Consortium and run through transcription factors for more reliable For more information, please visit us at the same internal validation program and consistent ChIP results. To learn www.activemotif.com/lowcellcs. used by our ChIP-Seq Services group. more, visit www.activemotif.com/tagchip.

Analyzing low abundance targets For a complete, up-to-date list of Increasing DNA binding site resolution (transcription factors) available antibodies, please visit us at For researchers working with tran- Performing ChIP-Seq on transcription www.activemotif.com/chipabs. scription factors, another common factors is often problematic due to the issue encountered with ChIP, aside relative low abundance of these proteins *For ENCODE Consortium guidelines for antibody validation, from low abundance of protein, is please refer to Landt S.G. et al. (2012) Genome Res. 22, and quality of available antibodies. To 1813–1831. the poor resolution of binding sites. address this, scientists at Active Motif Traditional ChIP-Seq methods only have developed the ChIP-IT® High Sensitivity Kit, the most sensitive ChIP kit available on the market. The kit uti- Tag-ChIP-IT Identifies Estrogen Receptor (ER) Binding Motifs lizes specially formulated reagents for preparation of high-quality chromatin ER Motif Identified Sample from minimal amounts of cells or tis- sue (1000 cells for abundant targets and 50,000 cells for low abundance AM-tag ER#1 transcription factors per reaction; the chromatin preparation module is also available separately as the High AM-tag ER#2 Sensitivity Chromatin Preparation kit). During the immunoprecipitation

(IP) reaction, low-background protein Figure 2: ER cDNA was cloned into pAM_1C Empty Vector and transiently transfected into cells. Cells were induced with estradiol G agarose beads and an antibody and chromatin was harvested and Tag-ChIP performed using the Tag-ChIP-IT Kit. Following cross-link reversal, enriched DNA was blocker reduce non-specific binding. submitted for next-generation sequencing. Data was compared to published anti-ER antibody ChIP-Seq results using the same cell Specialized ChIP buffers enhance line and induction conditions. ChIP-Seq data shows the same ER peak profile with the AM-tag ChIP as endogenous ER. Detected enrichment and reduce non-specific binding sites were further evaluated for binding motifs. Results show the ER motif was identified in both Tag-ChIP-IT samples.

[email protected] 7 CHROMATIN IMMUNOPRECIPITATION

applied across samples and antibodies antibody conjugates or an immunose- Active Motif’s to eliminate bias and reveal any real lection matrix and includes specialized ChIP-exo or obscured biological differences. buffers that prevent carryover of ChIP normalization can easily be primary-to-secondary immunoprecip- ChIP-seq implemented by integrating our Spike-in itation reactions. To learn more, visit reagents into your existing ChIP protocol. www.activemotif.com/rechip. ChIP-chip For more complete information, go to www.activemotif.com/spikein. To learn Active Motif’s recent addition to it’s about custom Spike-in Services, visit suite of custom services is RIME (Rapid www.activemotif.com/services-normalize. Immunoprecipitation Mass Spectrom- etry of Endogenous Proteins). RIME What interactions are occurring at resolves the complex process of gene

-300 0 300 my promoter of interest? regulation by enabling the capture When performing ChIP experiments, it and identification of interactomes, or Distance from motif (bp) is often useful to analyze co-occupancy the associated protein networks, of an Figure 3: ChIP-exo achieves significantly higher resolution of proteins or epigenetic modifications endogenous protein of interest. Gene of DNA binding sites than traditional ChIP methods. at a given genomic locus. Active Motif’s regulation research is often over- Re-ChIP-IT® Kit is a Sequential ChIP simplified by solely focusing on one (Seq-ChIP, Re-ChIP) procedure in regulatory factor. In reality, differen- resolve DNA binding sites within ≥200 bp when the average DNA binding site which ChIP samples are subjected to tial gene expression is the result of length of individual transcription factors sequential rounds of immunoprecipi- the highly orchestrated and complex tends to be in the region of 6-10 bp. tations using two different antibodies interactions of chromatin modifiers, To address this, Active Motif offers the to determine whether proteins or epigenetic modifications, coactivators ChIP-exonuclease (ChIP-exo) Kit con- epigenetic modifications co-occupy and repressors, and other regulatory taining an optimized and streamlined the same genomic region. The Active elements with chromatin. The RIME protocol for high-resolution genome- Motif Re-ChIP-IT Kit utilizes the same service includes immunoprecipitation wide mapping of transcription factor magnetic bead-based protocol used of a target protein from cross-linked binding sites. ChIP-exo is a modified in our ChIP-IT Express Kits to provide cell extracts followed by ChIP-Seq, ChIP-Seq approach that enables a streamlined Re-ChIP protocol for mass spectrometry and data analysis. researchers to identify protein-DNA sequential ChIP analysis. Unlike other binding sites at a resolution of 20-95 Seq-ChIP methods, the Re-ChIP-IT To learn more about our RIME Services, base pairs, refining the ability to identify method eliminates the need for direct visit www.activemotif.com/rime. DNA motifs more precisely and distill How can ChIP aid in genome editing? genome-wide protein binding profiles ChIP-Seq Normalization Workflow for studies of mutational or SNP CRISPR/Cas9 genome editing has effects on transcription factor binding gained prominence in genetic research sites (Figure 3). For more information, Experimental Spike-in and has expanded its capabilities in so Chromatin Chromatin go to www.activemotif.com/chipexo. many ways, providing a simple method Antibody of Spike-in to potentially genetically modify interest Antibody plants, eradicate viruses, and identify How do I normalize for sample bias? cancer genes. However, there are ChIP-Seq is a multi-step process in unintended consequences of this which disparities caused by sample technology, mainly issues with Cas9 loss during immunoprecipitation and nuclease specificity in creating double- Chromatin Immunoprecipitation library preparation, uneven sequencing stranded breaks leading to off-target read depth or technical variation can effects. To enable you to assess Cas9 Sequencing lead to results that are difficult to specificity, Active Motif’senChIP Kit interpret. To overcome this challenge, (Engineered DNA-binding molecule- Active Motif has developed a ChIP mediated Chromatin Immunopre- Map to experimental Map to rosophila Normalization Spike-in strategy for genome genome cipitation) offers a modified ChIP normalization of ChIP qPCR and assay that utilizes the CRISPR/Cas9 ChIP-Seq data to reduce the effects Normalize sample Normalize rosophila system to target a specific DNA locus tag counts by same ratio tag counts across samples of technical variation and sample for immunoprecipitation. Using the processing bias. This strategy can be enChIP Kit for your genome editing

8 www.activemotif.com CHROMATIN IMMUNOPRECIPITATION

chromatin from primary cells for ChIP RIME Service Workflow for Identification of Endogenous Interactomes analysis and next-generation sequenc- ing. For more information on the ChIP-IT FFPE and PBMC kits, please visit us at www.activemotif.com/ffpe or www.activemotif.com/pbmc. Sonicate Cross-link cells Isolate nuclei Analyzing chromatin-associated RNA For researchers interested in the role Identify binding of RNA in regulation of the genome, sites by ChIP-Seq Active Motif has adapted the magnetic bead-based method used in our ChIP-IT Immunoprecipitate target Express Kits for the development of Identify interacting the first-of-its-kind kit for RNA-ChIP. proteins by mass The RNA ChIP-IT® Kit is specifically spectrometry designed to study RNA-protein inter- actions in a chromatin context. RNA ChIP-IT’s innovative fixation method is experiments enables you to biologically to harvest valuable biological information superior to existing RIP technologies validate each target sequence for from clinical samples, many of the com- that use native nuclear extracts because specificity. Sequences demonstrating mon clinical sample types are primary fixation preserves chromatin interac- off-target effects can be excluded, tissues that present a challenge either tions, taking into account the dynamic saving valuable time and resources. because of the difficulty in extracting chromatin environment of the RNA- high-quality chromatin from difficult- protein complexes of interest. For more, To learn more, go to page 10 or visit us to-lyse cells, very limited amounts of visit www.activemotif.com/rnachip. at www.activemotif.com/enchip. sample, or highly degraded material. Active Motif provides the ChIP-IT® FFPE ® Can I also interrogate the DNA and ChIP-IT PBMC assays to finally en- For a complete list of available methylation status of my ChIP DNA? able researchers to perform ChIP anal- For those interested in DNA methyla- ysis of primary tissue samples. These ChIP products & services, tion profiles of their genomic region of ChIP assays are the only commercially please visit us at interest, Active Motif’s ChIP-Bisulfite- available assays specifically developed www.activemotif.com/chip Sequencing (ChIP-Bis-Seq) Kit offers and optimized to extract high-quality a method to directly interrogate both chromatin and DNA methylation status within the same sample. ChIP-Bis-Seq Product Format Catalog No. combines a ChIP procedure for target- ® specific enrichment of protein-bound ChIP-IT Express Kit 25 rxns 53008 or modified chromatin with bisulfite ChIP-IT® Express Enzymatic Kit 25 rxns 53009 conversion and sequencing (Bis-Seq) ChIP-IT® High Sensitivity Kit 16 rxns 53040 to allow single nucleotide resolution DNA methylation profiles of ChIP- High Sensitivity Chromatin Preparation Kit 16 rxns 53046 enriched chromatin. This approach Tag-ChIP-IT® Kit 16 rxns 53022 is ideal for genome-wide studies of ChIP-exo Kit 12 rxns 53043 the interactions between DNA meth- ylation and chromatin features, such Spike-in Antibody 50 µg 61686 as in assessing allele-specific DNA Spike-in Chromatin 10 µg 53083 methylation variances (e.g. imprinting, Re-ChIP-IT® Kit 25 rxns 53016 X-inactivation), or in evaluating global gene regulatory effects. To learn more, ChIP-Bis-Seq Kit 10 libraries 53048 visit www.activemotif.com/chip-bis-seq. ChIP-IT® FFPE Kit 16 rxns 53045 ChIP-IT® PBMC Kit 16 rxns 53042 Difficult-to-ChIP clinical samples In disease research, when attempting RNA ChIP-IT® Kit 25 rxns 53024

[email protected] 9 ChIP & CRISPR/Cas9 GENE EDITING

NEW Determine CRISPR/Cas9 Specificity Using enChIP

enChIP* is a modified chromatin immunoprecipitation (ChIP) assay that utilizes the CRISPR/Cas9 system to target a specific DNA locus for immunoprecipitation. Using the enChIP Kit enables you to biologically validate each target sequence for specificity. Sequences demonstrating off-target effects can be excluded from genome editing experiments, thereby saving valuable time and resources.

*Patent number: WO2014/125668

How does enChIP work? enChIP Kit ValidatesenChIP Specificity Kit Validates gRNA Specificity Figure 1: Two gRNAs were designed targeting Active Motif’s enChIP Kit (Engineered different 20 nt sequences within a 500bp Target Site region of a CTCF binding site on chromosome DNA-binding molecule-mediated CTCF Chromatin Immunoprecipitation) 19. Each gRNA sequence was cloned into ChIP-Seq the pAM_dCas9 vector and transfected into utilizes the CRISPR/Cas9 system to HEK293T cells. A ‘No gRNA’ negative control direct a guide RNA (gRNA) to a specific was also performed. Chromatin was prepared genomic locus for immunoprecipitation. gRNA2 and immunoprecipitated according to the The gRNA is co-expressed with an enChIP Kit. Enriched DNA was analyzed by NGS and background was subtracted using enzymatically inactive form of the gRNA1 the ‘No gRNA’ control. Data was compared to Streptococcus pyogenes Cas9 endo- ChIP-Seq data for CTCF in the same cell line. nuclease (dCas9). The dCas9 protein No gRNA Results show gRNA1 had a strong peak and contains Active Motif’s unique AM-tag specific binding at the target location. Data for sequence that was designed specifi- gRNA2 revealed a large number of off-target cally for high sensitivity enrichment binding events outside of the target location. This confirms that enChIP provides biological in ChIP. Following transfection of the validation for CRISPR/Cas9 specificity. gRNA and AM-tagged dCas9, cells are formaldehyde fixed, lysed, and the chromatin is fragmented by sonication. sequences bound by the gRNA/dCas9 What’s in the box? A monoclonal antibody directed against complex. Following immunoprecipita- The enChIP Kit contains chromatin the AM-tag is used to enrich for genomic tion, purified DNA can be analyzed preparation reagents, enChIP buffers, by qPCR or NGS to evaluate binding AM-tag monoclonal antibody, protein G specificity or chromosomal looping. magnetic beads and DNA purification enChIP Kit Highlights reagents for 16 enChIP reactions. The For more information, please visit us gRNA and dCas9 expression vectors • Identify off-target binding events at www.activemotif.com/enchip. are sold separately. • Evaluate chromosomal looping

• Versatility to use your own gRNA Product Format Catalog No. constructs or clone into one of Active Motif’s vectors enChIP Kit 16 rxns 53125 pAM_gRNA Vector 10 µg 53121 • Utilizes AM-tag specifically designed for use in ChIP pAM_dCas9 Vector 10 µg 53122 • High sensitivity assay detects pAM_gRNA_CTCF Vector (Positive control) 10 µg 53123 sequences present at only 1 or pAM_dCas9_CTCF Vector (Positive control) 10 µg 53124 2 genomic locations FuGENE® HD Transfection Reagent 0.2 ml 32042

10 www.activemotif.com GLOBAL DNA METHYLATION

Quantify Global Changes in 5-mC & 5-hmC in Normal and Diseased Samples The cancer epigenome is marked by alterations in a myriad of epigenetic features, including DNA methylation, nucleosome positioning and histone post-translational modifications. These changes influence gene regulation and can serve as signatures of cancer. To better understand how changes in DNA modifications influence normal and diseased states, Active Motif offers simple, easy-to-use assays to perform comparative studies of global changes in 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC).

Global DNA methylation & disease enzymatically digested to create LINE-1 Quantify changes in global 5-hmC levels In mammals, DNA methylation is fragments. The DNA is then hybridized Using Active Motif’s Global 5-hmC characterized by the covalent addition with a LINE-1 consensus probe. Quantification Kit, hydroxymethylcytosine of a methyl group within the cytosine Following capture onto a 96-well plate, levels can be quantified from as little of a CpG dinucleotide. These CpG a 5-mC antibody and colorimetric as 20-50 ng of genomic DNA. This sites are often found within repetitive detection reagents are used to generate easy-to-use plate-based assay offers regions and are normally methylated a signal that is easily measured with a higher throughput quantification using to repress transcription. In cancer, microplate reader. The assay includes less input material than mass hypomethylation of these repetitive DNA standards for 5-mC quantifica- spectrometry and can detect as little elements is observed. Likewise, the DNA tion and can detect as little as 0.5% as 0.02% 5-hmC with the included methylation variant 5-hmC is known to methylcytosine (Figure 1). DNA standards (Figure 2). play a role in transcriptional regulation and embryonic development and may Global DNA Methylation – LINE-1 Global 5-hmC Quantification also serve as a prognostic indicator in 0.9 2.5 0.8 certain cancers and neurodegenerative Global 5-hmC ELISA 0.7 disorders. Hydroxymethylcytosine 2 Mass Spectrometry 0.6 results from the oxidation of 5-mC 1.5 0.5 by the Ten Eleven Translocation (TET) 0.4

1 % 5-hmC enzymes. Because of their importance OD 450nm 0.3 in biological processes and disease, 0.5 0.2

0.1 methods to accurately quantify global 0 Blank 0% 1.3% 2.6% 3.9% 6.6% 9.9% 13.1% HeLa HeLa 0 5-mC and 5-hmC are highly warranted. + Aza mouse mouse mouse mouse mouse human human human % 5-mC of total cytosine content (100 ng/well DNA) brain heart kidney liver lung brain kidney liver

Quantify changes in global 5-mC levels Figure 1: The Global DNA Methylation – LINE-1 Kit was Figure 2: The Global 5-hmC Quantification Kit was used Active Motif’s simple, plate-based used to compare the methylcytosine levels between two to determine the % 5-hmC in genomic DNA isolated Global DNA Methylation – LINE-1 Kit genomic DNA samples. Assay results show a decrease from mouse and human tissues. Genomic DNA was in 5-mC levels resulting from treatment of HeLa cells tested at 20 ng/well for human brain and 50 ng/well for uses a consensus sequence within the with 5-azacytidine (Aza), a DNA methyltransferase inhibitor. all other DNA samples. The % 5-hmC was determined human repetitive Long Interspersed The % 5-mC is calculated using the included DNA standards. using the included DNA standards. Results were Nucleotide Element 1 (LINE-1) as a Methylcytosine levels of the DNA standards are displayed compared to mass spectrometry data obtained from 500 ng surrogate readout for global 5-mC as a percentage of total cytosine content. of the same DNA samples. Results show the Global levels. First, genomic DNA is 5-hmC Kit provides equivalent quantification using only a fraction of the starting material.

COMING SOON: Product Format Catalog No. Global LINE-1 Kit Global DNA Methylation – LINE-1 Kit 1 x 96 rxns 55017 for MURINE samples Global 5-hmC Quantification Kit 1 x 96 rxns 55018

[email protected] 11 miRNA TARGET IDENTIFICATION

A Genome-wide 3’UTR Collection for High-throughput Functional Screening of miRNA Targets To enable researchers to study microRNAs (miRNAs) using 3’UTR-reporter assays on a high-throughput scale, we have created a genome-wide collection of 12,000 human 3’UTR luciferase reporters in the highly-optimized LightSwitch™ Luciferase Assay System. The assay system is ideal for performing miRNA target validation and assessing the functional impact of miRNA-3’UTR interactions. Combined with our large collections of miRNA Mimics & Inhibitors, you have everything needed to study miRNA-3’UTR interactions, validate miRNA targets, and measure RNA stability and the functional impact of miRNAs on a gene-by-gene basis.

LightSwitch™ 3’UTR reporter throughput scale, we have created a miR-122 mimic (Catalog No. MIM0136) collection and assay system genome-wide collection of 12,000 or a non-targeting control (Catalog Identification and validation of miRNA human 3’UTR luciferase reporters in No. MIM9001). Luminescence for targets can be a complicated task. the highly optimized LightSwitch 58/142 (40.8%) of the predicted There are several alternatives available, Luciferase Assay System (Figure 1). targets was significantly different in starting with numerous target prediction the mimic co-transfection compared methods and continuing with different Luciferase-based screening to to the non-targeting control (p<0.05) biological procedures that encompass identify miR-122 targets (Figure 2). Thus, not only did this reporter-based screenings, shotgun The LightSwitch system represents screen identify numerous novel targets proteomics and Ago2-based immuno- a sensitive, specific and economical of miR-122, but we also found several precipitation methods. 3’UTR-reporter means to probe miRNA-UTR inter- targets with more potent interactions assays have become an important actions and functionally validate with miR-122 than previously observed. component of thorough miRNA target miRNA targets. To demonstrate, we These studies exemplify how our studies because they provide func- performed a high-throughput screen genome-wide library of human 3’UTR- tional evidence for, and quantitate to identify new targets of miR-122. luciferase reporter constructs enables the effects of, specific miRNA-3’UTR Target predictions were obtained researchers to screen thousands of interactions in a cell-based system. from commonly used sources such as potential miRNA targets and under- Thus, to enable researchers to leverage Miranda and Target Scan. Individual stand the roles of miRNAs in a single 3’UTR-reporter assays on a high- 3’UTR luciferase constructs (Catalog experiment. To learn more, please visit No. 32011) were employed in co-trans- us at www.activemotif.com/ls-3utr. luciferase 3’UTR promoter miRNA mimic fection experiments with either an co-transfect or 3’UTR non-targeting control Reporter 1.5 Predicted 3´UTR targets

1.0 RISC complex 0.5

Hybrid 0.0 LUC-3UTR Transcript -0.5

-1.0 Positive control 2

-1.5 miRNA luciferase activity miRNA luciferase Positive control 1

-2.0 log2 ratio of targeting/non-targeting of targeting/non-targeting ratio log2

-2.5

Figure 1: Experimental design for an miRNA target Figure 2: A functional screen of 142 human 3’UTRs with predicted miR-122 sites in HT-1080 cells. 44 of the targets decreased functional screen using the LightSwitch System. significantly by t-test (p<0.05) and 24 were significantly repressed >2-fold.

12 www.activemotif.com ANTIBODIES FOR CANCER RESEARCH

Validated Antibodies Reveal Histone-mediated Effects on miRNA in Non-small Cell Lung Cancer Research has shown that dysregulation of microRNA (miRNA) function is associated with many cancers. Furthermore, epigenetic silencing via DNA methylation can alter miRNA expression. In a recent study led by the laboratory of Dr. Nobuya Ohishi at the University of Tokyo Hospital, researchers looked at miR-139, a known tumor suppressor in several cancers, to study its function in non-small cell lung cancer (NSCLC). Using chromatin immunoprecipitation (ChIP) and Active Motif antibodies to trimethylated marks on Histone H3, they suggest that H3K27me3-mediated silencing of miR-139 may represent a promising biomarker of lung cancer metastasis.

Histone methylation-mediated silencing lysine 4 (H3K4me3), a mark of active of miR-139 enhances invasion of NSCLC transcription, and lysine 9 (H3K9me3) MiR-139-5p (denoted thereafter as which, along with H3K27me3, represent miR-139) has recently been reported transcriptional repressors. Aided by to function as a tumor suppressor in Active Motif’s ChIP validated antibodies several types of human cancers, but its to all 3 marks, ChIP analysis showed Figure 1: Structure of the PDE2A genomic locus. The function in NSCLC and the mechanism no real change for H3K9me3 across locations of miR-139 and the ChIP primers are shown. of its suppression have not been studied all cell lines and a positive correlation in detail. In a recent publication, research between H3K4me3 and PDE2A. How- conducted in the laboratory of Dr. ever, H3K27me3 had a strong negative Nobuya Ohishi reveals that miR-139 is correlation, more so with NCI-H2170 epigenetically silenced by histone H3 cells with the lowest expression lysine 27 trimethylation (H3K27me3) of levels of PDE2A (Figure 2). To further its host gene PDE2A, and that tran- demonstrate this correlation, siRNA scriptional regulation is independent inhibition of EZH2 H3K27me3 methyl- of promoter DNA methylation. transferase led to an increase in PDE2A in cancer cells, leaving the For more detailed information, go to researchers to conclude, at least in www.ncbi.nlm.nih.gov/pubmed/26256448. part, that H3K27me3 represents a promising biomarker of lung cancer To support their findings, researchers metastasis. Figure 2: ChIP analysis of H3K4me3, H3K27me3, and first confirmed the suppression of the H3K9me3 in five lung cancer cell lines and normal human host gene PDE2A, and the association bronchial epithelial cells. IP/Input values were normalized For a complete up-to-date list of of expression levels with miR-139, by to a control locus (ACTB promoter). available antibodies, please visit us at measuring their expression across www.activemotif.com/abs. several lung cancer cell lines, as well rule out any dependencies between as NSCLC and adjacent normal lung histone and DNA methylation. This article is a summary of the research performed by Watanabe et al. at the University of Tokyo Hospital, Hongo, tissue, using RT-PCR. Bunkyo-ku, Tokyo, Japan, published in Cancer Medicine To form a complete picture of tran- (2015) 4,1573–1582. With coordinated expression between scriptional regulation, ChIP was per- This is an open access article under the terms of the Creative miR-139 and PDE2A established, they formed on histone H3 trimethylated at Commons Attribution License. then turned to chromatin immunopre- cipitation to look at histone methylation Product Format Catalog No. around the promotor of the PDE2A Histone H3K4me3 antibody (pAb) 100 µg 39915 short transcript (Figure 1) to determine Histone H3K9me3 antibody (pAb) 100 µl 39161 if H3K27me3 was silencing PDE2A. Bisulfite sequencing was also used to Histone H3K27me3 antibody (pAb) 100 µg 39155

[email protected] 13 RECOMBINANT PROTEINS

High Quality Bromodomain Proteins for Developing Robust Assays for Epigenetic Drug Discovery Bromodomain proteins play an integral role in the regulation of transcription and chromatin remodeling. Because BET (Bromodomain and Extraterminal domain) proteins have been shown to also regulate transcription of certain oncogenes, they are promising therapeutic targets for cancer. As a result, there has been an increase in focus on bromodomain proteins in drug discovery research that has led to the discovery of a class of small molecule BET inhibitors. Early successes of BRD4 inhibition has peaked interest in the therapeutic potential of other BET inhibitors. To support research efforts, Active Motif offers a comprehensive collection of recombinant bromodomain proteins.

BET family members are promising chromatin resulting in MYC downregulation. therapeutic targets JQ1 has also been shown to target BRD2 Active Motif Bromodomain Proteins The BET family of bromodomain proteins in hematologic malignancies and inhibit • Over 40 reader domains available has been shown to play a key role in the expression of BRD3 and BRD4 in Tamoxifen development of several kinds of cancer. resistant breast cancer cells, giving it the • Specifically designed for Drug discovery efforts have led to the ability to perform as a pan-BET inhibitor. epigenetic drug discovery identification of multiple small molecule • Custom & bulk orders available inhibitors that target BET proteins, and I-BET151 and I-BET762 are similar to several of these inhibitors currently have JQ1 in that they possess nanomolar • Lot specific data, including: advanced to clinical trials. affinity and show strong anti-proliferative - Activity by HTRF effects. I-BET151 also functions as a - Purity by SDS-PAGE BETs are a subclass of proteins that pan-BET inhibitor showing anti-tumor are characterized by two N-terminal activity in multiple myeloma, malignant bromodomains and one ET (Extra brain tumor and other murine models. Due to the preliminary success of these Terminal) domain. The ET domain functions I-BET762 is considered a more specific therapeutics in the treatment of cancer, as a protein binding motif and exerts inhibitor and first showed promise in there is much interest in manipulating atypical serine-kinase activity, while the NUT midline carcinoma (NMC). Phase I specific domain interactions as a novel two N-terminal bromodomains, also known clinical trials for I-BET762 are ongoing approach for identifying bromodomain as BD1 and BD2, recognize acetylated for NMC as well as refractory hematologic targeted drug therapies. However, lysines on histones and function to malignancies. there is still much unknown regarding recruit other BET proteins to chromatin. BET proteins and the individual BDs. Because the recruitment and targeting of serine-kinase and other activities by Bromodomains BETs to chromatin involve the concerted purity by SDS-PAGE interplay of several BET domains, it is BRD2 (71-194) BRD3 (306 - 416) BRD4 (44 -168) crucial that we understand the effects of kDa kDa kDa 55 55 55 40 existing inhibitors on individual domain 40 40 35 functions. 35 35 25 25 25 The quest for inhibitors of BET function One of the most widely studied BET 15 15 15 inhibitors is JQ1, a small molecule inhibitor of BRD4 with nanomolar affinity that shows promise across many BRD4 dependent cell lines. As an effective Figure 1: Purified recombinant proteins were run on an SDS-PAGE gel and stained with Coomassie blue. Coomassie anti-proliferative agent, JQ1 acts as a stains reveal that the recovered proteins are intact with little or no detectable degradation. All proteins were determined to have ≥ 98% purity. competitive binder, displacing BRD4 from

14 www.activemotif.com RECOMBINANT PROTEINS

15K BRD4 (44-168) Titration Phylogenetic Tree Diagram of the Human Bromodomain Family

10K

BRPF1

BRD1 HTRF Signal 5K

CREBBP

EP300 0 BRPF3 BRD2 0 50 100 150 200 250 300 BRD9 BRD3 Bromodomain conc. (nM)

BRD4 BAZ1A BRD4 (333-460) Titration BRD7 15K BRD3 KIAA1240 BRDT BRDT BRD2

10K ATAD2 BRD4

BRD8 BRDT BRDT HTRF Signal 5K BRWD1

TAF1 TAF1L PHIP PRKCBP1 BRWD3 TAF1 0 BAZ1B 0 50 100 150 200 250 300 TAF1L Bromodomain conc. (nM)

BAZ2B PCAF Figure 2: BRD4 (44-168) (top panel), and BRD4 CECR2 FALZ GCN5 (333-460) bromodomains (bottom panel) tested BAZ2A ZMYND11 by HTRF. Assay conditions for bromodomain (BRD) activity were as follows: 3.3 uM histone peptide H4K5/8/12/16(tetra-acetyl) was incubated with BRWD1 the protein indicated in reaction buffer containing TRIM33 50mM HEPES-NaOH pH 7.0, 0.1% BSA for 1 hour MLL BRWD3

SP110 PHIP at room temperature. Anti-FLAG antibody was TIF1 ASH1L used to detect the reaction products. TRIM28 TRIM66 SP100

PB1 (6)

Research and inhibitor screening has PB1 (3) SP140 markedly increased in recent years PB1 (1) suggesting a need for validated products SP140L PB1 (4) to support these efforts. SMARCA4 PB1 (2)

Active Motif has responded to this need SMARCA2 by providing the most comprehensive and PB1 (5) validated collection of reader domain pro- teins for use in the development of robust assays for bromodomain drug discovery.

Figure 3: Active Motif provides a comprehensive collection of high quality reader domain proteins for use in assay The most comprehensive reader domain development in early drug discovery. Bromodomain proteins, designated with purple dots, represent currently available collection available for drug discovery bromodomains in our collection. In addition, as part of our integrative solutions for epigenetic drug discovery, As a global leader in providing new and Active Motif also offers custom and bulk orders, as well as antibodies and inhibitors to these proteins. innovative products and services to enable epigenetics research, Active Motif offers a our recombinant proteins and enzymes platforms and unparalleled customer comprehensive portfolio of recombinant demonstrate the nM range activities support for our drug discovery partners. epigenetic enzymes and reader domains needed for use in identification of potent for drug discovery research (Figure 3). inhibitors. For more information and an up-to-date Our recombinant proteins are manufac- list of available proteins, please visit tured to meet the highest standards of At Active Motif, we have optimized our www.activemotif.com/proteins. purity (Figure 1) and activity required for protein production and testing to facilitate conventional drug discovery assays, such seamless integration of our recombinant as HTRF (Figure 2), mass spectrometry, proteins into drug discovery pipelines. We To inquire about AlphaLISA and Alphascreen. These assays strive to instill confidence in our proteins Custom and Bulk Orders are routinely incorporated as part of our by providing comprehensive application CALL: 877-222-9543 quality control process to ensure that data that is relevant to drug discovery

[email protected] 15 PRSRT STD US POSTAGE PAID PERMIT #94 SAN DIEGO, CA

NORTH AMERICA JAPAN EUROPE CHINA

Toll Free: 877 222 9543 Direct: +81 (0)3 5225 3638 GERMANY 0800/181 99 10 Hotline: 400 018 8123 Direct: 760 431 1263 Fax: +81 (0)3 5261 8733 UNITED KINGDOM 0800/169 31 47 Direct: +86 21 2092 6090 Fax: 760 431 1351 [email protected] FRANCE 0800/90 99 79 [email protected] [email protected] OTHER COUNTRIES, DIRECT +32 (0)2 653 0001 [email protected] Fax: +32 (0)2 653 0050 [email protected] Customer Service: [email protected]

Enabling Epigenetics Research