CHROMATIN IMMUNOPRECIPITATION Diagenode provides complete solutions for ChIP, facilitating workflows and ensuring consistent results in every experiment PAGE 2 DIAGENODE CHROMATIN IMMUNOPRECIPITATION
Complete solution for your ChIP
Whether you are experienced or new to the field of chromatin immunoprecipitation, Diagenode has everything you need to make ChIP easy and convenient while ensuring consistent data between samples and experiments. As an expert in the field of epigenetics, Diagenode is committed to providing complete solutions from chromatin shearing reagents, shearing instruments such as the Bioruptor® (the gold standard for chromatin shearing), ChIP kits, the largest number of validated and trusted antibodies on the market, and the SX-8G IP-Star® Compact Automated System to achieve unparalleled productivity and reproducibility.
INDUSTRY-LEADING CHIP KITS Diagenode offers a broad portfolio of extensively validated, high-quality ChIP kits. Choose the one that fits with your specific experimental needs( page 8).
CHIP-SEQ AND CHIP-GRADE ANTIBODIES Diagenode focuses exclusively on epigenetics, providing the largest selection of industry-leading validated antibodies with dedicated technical expertise and support. Our antibodies are the only ones on the market validated in-house for ChIP-seq. Additonally, Diagenode offers premium antibodies which have reached the highest level of validation from extensive in-house validation in combination with numerous collaborations with the EU community of epigenetic experts (page 22).
BIORUPTOR® SONICATION DEVICE The Bioruptor is now the industry’s standard and most cited tool for DNA shearing, chromatin shearing and cell lysis*, and is validated for Next-Generation Sequencing library preparation, ChIP, and DNA methylation studies (page 30).
AUTOMATED SOLUTIONS The SX-8G IP-Star® Compact Automated System is a simple yet robust automated bench-top instrument that standardizes different epigenetic applications (e.g. ChIP), delivers consistent data and increases your lab’s productivity (page 34).
* See list of selected publications on page 31
Innovating Epigenetic Solutions PAGE 3
About Epigenetics Table of contents
In 2003, U.S. National Human Genome Research Institute About Epigenetics (NHGRI) director Francis Collins declared, “The HGP has Epigenetic Modifications of Histones...... 4 been an amazing adventure into ourselves, to understand our own DNA instruction book, the shared inheritance of all What is ChIP? ...... 6 humankind.” Raw sequences of an estimated 30,000 genes Diagenode’s ChIP-seq workflow...... 7 coding for various cellular functions were identified. STEP 1: Chromatin Shearing Optimization Since that time, much of the focus in human molecular Chromatin Shearing Optimization kits ...... 8 biology has been on the study of gene regulation in response to intra- and extra-cellular stimuli. This led to one of the STEP 2: Sonication challenges in modern biology: understanding the processes Overview ...... 9 and mechanisms that allow a single cell to become a ® complete organism composed of hundreds of individual cell Bioruptor Gold standard for chromatin shearing . . . . 10 types, all of which still contain the same DNA. Researchers Efficient chromatin shearing with the Bioruptor® Plus . . 11 soon discovered that chromatin could be likened to an The shearing device of choice...... 12 “instruction book” which needs to be opened first so that Bioruptor® Models...... 13 the words (i.e. the genes), can be read and interpreted. The epigenetic regulation of gene expression refers to STEP 3: Immunoprecipitation this phenomenon. The term epigenetics defines what is Antibodies...... 14 happening on the chromatin, the physical support of genes. Manual ChIP kits...... 32 Epigenetics is also defined as the study of heritable changes Automated ChIP kits...... 37 in phenotype that are independent of changes in the DNA Automation ...... 40 sequence. “Epigenetic landscape” was a definition introduced by Conrad H. Waddington in the first part of the 20th century STEP 4: DNA Purification as “... the interactions of genes with their environment which IPure kit...... 44 bring the phenotype into being” to describe the phenomena connecting genotype to phenotype and describing the STEP 5: Library Preparation process of differentiation. MicroPlex Library Preparation kit...... 45 iDeal Library Preparation kit...... 46
About Diagenode...... 47
Diagenode Headquarters ...... Back Cover
© 2016 Diagenode SA and Diagenode Inc. All rights reserved. No part of this publication may be reproduced, transmitted, transcribed, stored in retrieval systems, or translated into any language or computer language, in any form or by any means: electronic, mechanical, magnetic, optical, chemical, manual, or otherwise, without prior written permission from Diagenode SA (hereinafter, “Diagenode”) . The information in this guide is subject to change without notice. Diagenode and/or its affiliates reserve the right to change products and services at any time to incorporate the latest technological developments. Although this guide has been prepared with every precaution to ensure accuracy, Diagenode and/or its affiliates assume no liability for any errors or omissions, nor for any damages resulting from the application or use of this information. Diagenode welcomes customer input on corrections and suggestions for improvement.
The trademarks mentioned herein are the property of Diagenode or their respective owners. Bioruptor® and IP-Star® are registered trademarks of Diagenode SA. Illumina is a registered trademark of Illumina Inc. Ion Torrent and Personal Genome Machine are trademarks of Life Technologies Corporation.
www.diagenode.com | PAGE 4 DIAGENODE CHROMATIN IMMUNOPRECIPITATION
In 1942, Waddington stated “... it is possible that an adaptive Three major levels of epigenetic changes are described response can be fixed without waiting for the occurrence today: of a mutation...” explaining the plasticity of epigenetic processes, and thereby framing their potential heritability, Chemical modifications at the level of the or long-term stability. nucleotides, which include DNA methylation and RNA interference Epigenetic machinery gives an organism with the molecular Modifications at the level of histones that include resources a quick response to environmental conditions post-translational change of histone proteins and with a steady modification in gene expression. the incorporation of histone variants Visual representation of the epigenetic landscape. ATP-dependent processes that regulate the access to nucleosomal DNA (nucleosome remodelling)
Epigenetic mechanisms are made of a highly complex and interlacing network, incorporating all three levels of possible epigenetic modifications. These mechanisms include multiple positive and negative feed forward and feedback pathways that span both the DNA and the histone level and the higher order of chromatin structure. A number of epigenetic modifications are found at both of these levels due to mechanisms which still remain poorly understood.
Epigenetic Modifications of Histones
Post-translational histone modifications affinity between the histone tail and the negatively charged DNA. Histones can become acetylated on lysine (K) (PTMs) residues. Histone acetylation are regulated by the histone acetyltransferases (HATs) and two functional categories of Due to their chemical properties, PTMs modify the histone deacetylases (HDACs): 1) eleven zinc-dependent condensation of chromatin and therefore the accessibility HDACs (class I, IIa, IIb, and IV HDACs) and 2) the sirtuins of DNA to transcriptional machinery. The majority of histone (seven class III NAD -dependent HDACs). Functional impact PTMs arises on the NH2-terminal tail, although on the of some acetylateable residues is more substantial than COOH-terminal tail of the core histones. PTMs consist of others. For example, in yeast (S. cerevisae), H4K16 regulates acetylation, methylation, phosphorylation, ubiquitination, its own subset of genes, while other genes are co-regulated and sumoylation. PTMs can occur on all histones. by all of H4K12, K8, and K5, but not by K16. Histones are basic proteins that regulate the compaction of the chromatin. Histones consist of a globular histone 2. Methylation core and a NH2-terminal histone tail, protruding out of the rigid histone-DNA assemblage, called the nucleosome. This epigenetic modification has been shown to associate A nucleosome, the 10 nm thick primary structure of the with both transcribed and silenced genes. Histones can be chromatin is made of an octamer of the four core histones methylated on both lysine and arginine (R) residues. Lysine H2A, H2B, H3, and H4 in duplicates, around which 147 residues can be mono-, di-, and trimethylated and arginine base pairs of DNA are wrapped. Nucleosomes are bonded residues can be mono- and dimethylated. Effector proteins, together by the linker histone H1, which is not part of the such as transcriptional co-activators, recognize mono-, di-, nucleosome as such. and trimethylated epitopes with different affinities. Thus it is most likely that the various degrees of histone methylation influence gene expression regulation (e.g. trimethylated 1. Acetylation H3K4 is exclusively associated with expressed genes Histone acetylation has been correlated to transcriptional whereas monomethylated H3K4 has been associated with activation. Acetyl group addition neutralizes the positive both expressed and repressed genes). charge of the -amino group of the lysine, which decreases
Innovating Epigenetic Solutions PAGE 5
4. Ubiquitination Whereas H2A ubiquitination is considered repressive, H2B ubiquitination has been associated with both active
Chromosome and repressed genes.Histone ubiquitination occurs in the attachment of the 76-amino acid protein ubiquitin to the histone core proteins H2A and H2B. H2A and H2B ubiquitination have different consequences on the transcription of the gene on which they are located.
H3K9me3 H2B ubiquitination is thought to be a condition for H3 H3K27me3 H4K20me3 methylation, but H2A inhibits this methylation. Thus, histone methylation and H2A / H2B ubiquitination frequently interact, HETEROCHROMATIN “Silent” particularly through H3K4 di- and trimethylation.
Nucleosome 5. Sumoylation Histone tail Histone sumoylation is transcriptional repression by Histone post-translational modification prevention of histone acetylation and by interaction with other repressor complexes. Histone octamer
H2B H2A SUMO proteins are comparable to ubiquitin, approximately H4 H3 100 amino acids long and added to their targets by specific ligases reversibly (proteases).
Histone variants
Sequential and structural variations of the core histones EUCHROMATIN result in histone variants. These variations can be the “Active” replacement of a few amino acids or of a large group of amino H3K4me3 H3K36me3 acids in the histone tails of the globular central domains. H3K79me3 Histone acetylations DNA double helix Histone variants can be integrated into specific regions of the genome throughout the cell cycle while the four core histones are incorporated into the nucleosomal structure exclusively Histone methylation is controlled by enzymes like histone during DNA replication. Thus, integration of histone variants methyltransferase (HMTs) specific to K and R residues as well can locally alter gene regulation. Since the core histones and as by histone demethylases (HDMs) such as peptdidylarginine their variants are structurally similar, they can be exchanged deiminase (PADI), lysine-specific demethylases (LSDs), and through an unknown mechanism. jumonji-C domain containing histone demethylases (JMJCs). A phenomenon called “histone eviction”, resulting in single It is the precise position of the histone methylation within histone or entire histone octamers displacement has a a given genetic region that determines the overall effect on profound influence on the compaction of chromatin and gene transcription. For the same gene, H3K9 methylation consequently on gene transcription. within the coding region of a gene has been found to correlate with gene expression, whereas the same modification in the Nucleosome Remodeling promoter region is associated with inactive transcription. Nucleosome remodeling is a process that creates transformations in the structure of chromatin and which 3. Phosphorylation requires a net energy input (carried out by enzymes that are Histone phosphorylation is most commonly associated with catalytically dependent on ATP as an energy source). transcriptional activation. Histone phosphorylation occurs on With ATP as a substrate, they facilitate the sliding of serine (S), threonine (T), and tyrosine (Y) residues because the histone octamers to adjacent DNA sequences and thereby negative charge of the phosphate group creates a repulsive the enrichment of accessible DNA on the surface of force between the histone and the negatively charged DNA. nucleosomes. Enzymes contain the SWI/SNF subunit of Histone phosphorylation is regulated by protein kinases ATPases (in eukaryotes). (PKs) and protein phosphatases (PPs).
www.diagenode.com | PAGE 6 DIAGENODE CHROMATIN IMMUNOPRECIPITATION
What is ChIP? ChIP workflow ChIP may seem intimidating but with the right tools can STEP 1. Chromatin preparation certainly be mastered. The principle of ChIP is simple, Cross-linking of chromatin and cell lysis involving the selective enrichment of a chromatin fraction containing a specific antigen. Antibodies that recognize a protein or protein modification of interest can be used to determine the relative abundance of that antigen at one STEP 2. Chromatin shearing or more locations in the genome in vivo. ChIP is extremely versatile as it may be used to compare the enrichment of a protein modification at different loci, map a protein modification across a locus of interest or quantify a protein modification on an inducible gene over a time course. ChIP is an ideal method for researchers interested in learning how STEP 3. Magnetic IP Cromatin fragments with a specific protein interacts with the genome in its “natural”
associated proteins state.
There are two general procedures for carrying out ChIP experiments: native ChIP (N-ChIP) and cross-linking ChIP (X-ChIP). The choice between N-ChIP and X-ChIP depends on your experimental aims and the starting material used: Immunoprecipitate with antibody specific to protein of interest by N-ChIP: Native chromatin is used as the substrate, which using magnetic bead (protein A or G) means that proteins are not cross-linked to the DNA. Fragmentation of the chromatin is achieved by micrococcal nuclease digestion, resulting in a nucleosome based resolution. N-ChIP is restricted to proteins that are very tightly associated with chromatin, typically limiting this Magnetic separation and washes type of ChIP to histones and their modifications (O’Neill and to eliminate unbound material Turner, 2003).
X-ChIP: Proteins are cross-linked to the DNA. Cross-linking is typically achieved using formaldehyde, and chromatin is randomly fragmented by sonication. As the proteins are cross-linked to the DNA, a broad range of chromatin associated factors can be analyzed.
STEP 4. DNA purification
STEP 5. Library preparation
Next Gen Seq Microarray qPCR
Protein of interest DNA
Other protein Magnetic bead
Antibody Magnet
Innovating Epigenetic Solutions PAGE 7
Bringing it all together: Diagenode's ChIP-seq workflow
1 CHROMATIN PREPARATION 2 DNA SHEARING
• Chromatin Shearing Optimization kit (Bioruptor® Sonication) (Low SDS, Medium SDS and High SDS) • Increased Reproducibility • Automated & High - Throughput EP 1 ST • No “Foaming” S TE P • No Risk of Contamination 2 IP-STAR® COMPACT AUTOMATED SYSTEM
NEXT GENERATION SEQUENCING
Bioruptor® Pico
3
P E T S
ST EP 5 4 3 MAGNETIC IP STEP 5 LIBRARY PREPARATION • Auto True MicroChIP kit • Auto iDeal ChIP-seq kit for Histones • MicroPlex Library Preparation Kit • Auto iDeal ChIP-seq kit for Transcription Factors (50 pg - 5 ng input material) 4 DNA PURIFICATION • Auto Plant ChIP-seq kit • iDeal Library Preparation Kit • Auto IPure kit v2 • True MicroChIP kit (5 ng 1 µg input material) (magnetic purification) • MicroChIP DiaPure columns • iDeal ChIP-seq kit for Histones • iDeal ChIP-seq kit for Transcription Factors • Plant ChIP-seq kit
Figure 1. Diagenode provides a full suite of manual and automated solutions for ChIP experiments For Step 1, we offer products to isolate nuclei and chromatin. Step 2 describes reproducible sample shearing with the Bioruptor® product line. In Step 3, 4 and 5 the Diagenode IP-Star® Compact provides error-free, walk-away automation for all your immunoprecipitation, DNA purification and library preparation needs.
www.diagenode.com | Innovating Epigenetic Solutions PAGE 8 STEP 1 CHROMATIN SHEARING OPTIMIZATION Optimization kits Chromatin Shearing DIAGENODE buffers from Corresponding to shearing Allows for shearing of Nuclei isolation SDS concentration Cat. No. Get consistent results from different samples andruns Obtain perfect fragment sizefor highqualityChIP Choose akitfor your specific experimental needs CHROMATIN IMMUNOPRECIPITATION cells on a1.5%agarose gel. analyzed are DNA of µl 10 RNase. with treated were the Bioruptor and AA-001-0100) No. (Cat. kit Optimization Shearing Chromatin the using sheared been has cells K562 of Chromatin subsequent ChIP Perfect fragment sizefor Chromatin Shearing Kit iDeal ChIP-seqkitfor 100 millioncells C01020010 Low SDS Histones < ® 0.1% Al samples All . Yes CHROMATIN SHEARINGEFFICIENCY ANTIBODY PERFORMANCE INCHIP EPITOPE ACCESSIBILITY
Chromatin Shearing Kit iDeal ChIP-seqKitfor Transcription Factors 100 millioncells C01020013 Low SDS 0.2% Yes excessive reaction volume, limited cell amounts available, etc.) (e.g. workflow your with ChIP, compatible be always to not might prior this dilute to possible is it Although ChIP. during binding antibody the alter or epitope the of inaccessibility to lead for might carefully concentrations Excessive chosen function. to be assay your to has SDS of concentration right the Therefore, shearing. chromatin successful for are SDS) essential (e.g. detergents that described widely been has It Key to successful chromatin shearing Higher detergent concentration may be beneficial with beneficial be difficult cell typesasitincreases theshearingefficiency. may concentration detergent Higher When you needahighSDSconcentration: When you needalow SDSconcentration: Limited cell amounts (epitopes are inaccessible) concentrations because ofprotein denaturation Antibodies maybeinhibited for IP-Star Maximum reaction volume SDS concentration) priorto ChIP avoid thedilution ofyour chromatin (to reach lower
Chromatin Shearing Kit HighCell# ChIPkit 100 millioncells ® Medium SDS CompactAutomated System) C01020011 0.5% Yes are available andyou wishto cannot beexceeded (e.g.
byhigherSDS Chromatin Shearing Kit True MicroChIP kit LowCell# ChIPkit 100 millioncells C01020012 High SDS 1% No
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Overview Bioruptor® Sonication Some of the key design features of the Bioruptor® are the laboratory friendly format, ability to use many sample Bioruptor® ensures success for a variety of tube types in a water bath-based rotor, and flexible power applications by using state-of-the art ultrasound controls. The walls of the water bath reflect the ultrasound technology to disrupt, disperse, or shear a variety of waves in a random but reproducible pattern. The samples sample types for biological, chemical, pharmaceutical in the adaptor are rotated through the ultrasound field to and industrial applications. The Bioruptor® is widely expose each sample to the same level and intensity of energy. used in biological settings and has proven success This novel technology enables a wide range of applications for efficient, reproducible sonication required for for superior yields and quality. applications such as chromatin shearing, DNA and RNA shearing, and cell and tissue disruption. Researchers have confirmed the Bioruptor® as an optimal shearing system, surpassing industry standards with high yields of superior quality material, as exemplified byover 2,000 publications.
Application versatility Chromatin shearing (ChIP-seq, ChIP-qPCR, ChIP-chip, ChAP, ChIRP etc.) DNA and RNA shearing (NGS library preparation, RNA-seq library preparation, MeDIP, MeDIP-seq, MethylCap, bisulfite conversion, etc.) Cell and tissue disruption (bacteria and yeast cell disruption, western blot, RIP, RIP-seq, The Bioruptor® uses ACT (Adaptive Cavitation Technology) MIRA-chip analysis, fractionation, etc.) to create focused mechanical stress to shear chromatin, Other biological applications (Mitochondria DNA or RNA or to lyse cells. The ultrasound waves pass disruption, cell dissociation, plant cell through the sample, expanding and contracting the liquid. transformation, etc.) During expansion, negative pressures pull the molecules away from one another and form a cavity or bubble. The Chemical, pharmaceutical and industrial bubble continues to absorb energy until it can no longer applications (Dispersion, emulsification, sustain itself and then implodes, producing intense focused homogenization, sonochemistry, liposomes shearing forces, which disperses or breaks biomolecules. preparation, etc.)
Bioruptor®
www.diagenode.com | Innovating Epigenetic Solutions PAGE 10 STEP 2 SONICATION DIAGENODE Reid G,Costa IG,Wagner W. factor TH, Brümmendorf V, Benes JoussenS, I, Charapitsatranscription S, Hummel specific at sites. methylation binding DNA with and Replicative senescence is associated with nuclear reorganization 3627-43 6: 28) BH. Howard AH, DeCherney JH, Segars J, Graham A, Ignezweski JC, Mullikin S, Hartley S, Gravina VR, Russanova B, gene in changes density.GC 3’-end and methylation human age-relatedbody gene expressionto links in cells sequencing granulosa transcriptome ovarian and methylome 15) DNA Jan (2015 Res J. Huehn Acids M, Lochner T, Nucleic Sparwasser J, Pezoldt L, Groebe IV, vivo differentiation. in Th17 during signature epigenetic unique a of Development MethylCap-seq Petronis A,KlimašauskasS. Koncevi Kriukien CpG sites. of capture covalent by profiling unmethylome DNA Roa JC,Guerrero-Preston R. P, Guzman O, D, TapiaSidransky R, Irizarry W, E, Criekinge Van L, NesteVan S, Muñoz Soudry J, Perez M, C, Brait Michailidi P, Garcia J, P, Ribas Leal population. C, Ili Chilean MG, Noordhuis a L, in Maldonado ethnicity and status with HPV associated neoplasia, cervical in methylated frequently proteinFK-506-binding and (ZNF516) 516 promoters(FKBP6) 6 protein finger Zinc reveals profiling methylation Genome-wide Li DF,ZhangJA. L, Xiao WJ, Jiang WangLF, Q, Shi Qin X, RH, Song FS, Muhali TT, analysismethylation DNA Genome-wide Graves’in disease. MeDIP-chip Eeckhoute J. P, Lefebvre B, Staels G, Salbert P, Froguel E, Durand S, Avner classes. enhancer distinct functionally through adipogenesis during metabolism involved lipid growthinsulin/insulin-likeand in factorsignaling receptor proliferator-activated Peroxisome M. Hirst JF, Costello TD, Tlsty MA, MarraPJ, FarnhamSJ, T, Jones WangE, PlettnerZiv Y, P, Ma MooreA, MT, Mungall McManus D, R, Hu D, Neel JB, Cheng MJ, Hangauer Moksa H, C, O’Geen A, L, Hong Echipare RP, Heravi-Moussavi Nagarajan B, Davis I, A, Hussainkhel Li M, Mingay D, M, Cheung G, Chu M, Robertson Griffith S, A, Cho B, Kamoh A, Tam A, Delaney A, Carles human the of breast. determinants transcriptional and Epigenetic DNA to of Helin K. leads induction J, Christensen OA, Bernard BT, cells Porse FO, and Bagger N, Rapin MT, enhancers hematopoietic active leukemogenesis. in of TET2 hypermethylation of Loss MeDIP-seq More than 2,000 paper citations. Only a small selection is given below. DNA methylationstudies Bioruptor Nat Commun (2015 Feb 18) 6: 6351 6: 18) Feb (2015 Commun Nat Gascard P, Bilenky M, Sigaroudinia M, Zhao J, Li L, L, Li J, Zhao M, Sigaroudinia M, Bilenky P, Gascard č CHROMATIN IMMUNOPRECIPITATION ė Genes Dev. (2015 May 1) 29: 910-22 29: 1) May (2015 Dev. Genes ius K, Li D, Wang T, Pai S, Ptak C, Gordevi , are , hr T Urbanavi T, Khare V, Labrie E, J Biol Chem (2014 Jan 10) 289: 708-22 289: 10) Jan (2014 Chem Biol J Hänzelmann S, Beier F, Gusmao EG, Koch CM, CM, Koch EG, Gusmao F, Beier S, Hänzelmann ® Genomics (2015 Jan 22) Jan (2015 Genomics Rasmussen KD, Jia G, Johansen JV, Pedersen JV, Johansen G, Jia KD, Rasmussen reference papers reference Oger F, Dubois-Chevalier J, Gheeraert C, C, Gheeraert J, Dubois-Chevalier F, Oger Yang BH, Floess S, Hagemann S, Deyneko S, Hagemann S, Floess YangBH, Nat Commun (2013 Jul 23) 4: 2190 4: 23) Jul (2013 Commun Nat Clin Epigenetics (2015 Jan 1) 7: 19 7: 1) Jan (2015 Epigenetics Clin Epigenetics. (2014 Feb 1) 9: 308-17 9: 1) Feb (2014 Epigenetics. č i ū γ t regulates genes genes regulates ė , Lapinait G, č ius J, Wang SC, Brebi P, P, Brebi (2015 Feb Feb (2015 ė Cai Cai A, A, Yu Yu
o al urn CI asy, rvdn otml yields, optimal frequentlyrequired Bioruptor applications.The ChIP forall providing assays, ChIP lengths,consistency.and Different fragment rangessize are current all for The chromatin shearing Bioruptor Allows parallel processing ofupto 12samples. High-throughput capability allow for microliter to milliliter quantities. holders sample – interchangeable volume sample flexible with Scales tubes. standard with workflow current into Fits Flexible format for DNAshearing,cell lysis andotherapplications. Besides chromatin shearing the Bioruptor sonication. of duration modify toprogrammed easily be can Cooling system maintains integrity ofsensitive samples. Temperature controlled Gentle ultrasound methodpreserves sample. Gentle ultrasound disrupts samples. Easy to program. Variable power range efficiently and evenly Flexible control guarantees bath No lysis water and shearing consistency. in ensuring tubes. energy of tubes distribution equal sample closed of Continuous rotation formation. with aerosol and cross-contamination contamination Prevents Rotation Bioruptor The Bioruptor ®
® is the is achieves highly consistent fragmentation ® -Goldstandard for chromatin shearing chromatin ® can also be used device of choice of device ®
PAGE 11
Optimizing sonication for even the most diffcult cells is easy with the Bioruptor®
Sonication should be optimized for each new cell type since cells can be different in their resistance to sonication. In addition, some cell lines, primary cells, lymphocytes, and fibroblasts are more difficult to shear than others. The Bioruptor simplifies the optimization process (Figure 1) as well as optimizing chromatin shearing from difficult cells (Figure 2).
5 cycles 10 cycles 15 cycles
Figure 1: HeLa cells were fixed with formaldehyde and chromatin was prepared according to Diagenode’s Chromatin Shearing Optimization Kit- Low SDS (Cat. No. C01020010). Samples were sonicated for 5-10- 15 cycles of 30” ON/30” OFF as indicated with Bioruptor® Pico using 1.5 ml Bioruptor® microtubes with caps (Cat. No. C30010016). A 100 bp ladder was loaded as the size standard.
A. MT-4 cells B. granulocytes C. Jurkat cells D. PBMC
5 10 5 10 MW 5 cycles 10 cycles MW cycles cycles MW cyclescycles MW 8 cycles
Figure 2: Successful chromatin shearing from “difficult” cells using the Bioruptor® Pico. MT- 4 (panel A) , granulocytes (panel B) Jurkats cells (panel C) and PBMC cells (panel D) were fixed with formaldehyde and chromatin was prepared accordingly to Diagenode’s Chromatin Shearing Optimization Kit- Low SDS (Cat. No. C01020010). Samples were sonicated for 5-10 cycles of 30” ON/30” OFF as indicated with Bioruptor® Pico using 1.5 ml Bioruptor® microtubes with caps (Cat. No. C30010016). A 100 bp ladder was loaded as size standard.
www.diagenode.com | PAGE 12 DIAGENODE CHROMATIN IMMUNOPRECIPITATION
The shearing device of choice for library preparation and chromatin preparation for ChIP
Fast, with small and large volume shearing (5 µl - 2 ml)
Optimized for:
> Next-Generation Sequencing (5 - 100 µl) > Chromatin shearing (10 µl - 2 ml) > RNA shearing > Protein extraction > FFPE DNA Extraction
• EASY TO USE AND INSTALL • SMALL AND LIGHT • TEMPERATURE CONTROLLED
1 2 3
Bioruptor® Pico (Sonication device 1 + Water cooler 2 + Single Cycle Valve 3
The Bioruptor® Pico is the shearing device of choice for sequencing applications, providing optimal yields, lengths, and consistency. Different fragment size ranges are frequently required for downstream applications (e.g. bridge amplification) for sequencing. Moreover, the Bioruptor® Pico is the optimal device for chromatin shearing for ChIP-seq applications. The Bioruptor® Pico can be easily programmed to modify duration of sonication for optimal fragmentation and produces:
• Simultaneous sonication of 12 samples • Cost effective solution • Compatible with all current Next-Generation Sequencing systems • Desired narrow size distribution crucial for sequencing accuracy • High yields of double-stranded DNA needed for effective sequencing results
LIFE TECHNOLOGIES RECOMMENDS THE BIORUPTOR® SYSTEM TO GENERATE DNA LIBRARIES FOR THE ION PERSONAL GENOME MACHINE (PGMTM) AND THE PROTON™
Innovating Epigenetic Solutions A flexible selection of Bioruptor® models suits your needs
Bioruptor® Standard Bioruptor® Plus Bioruptor® Pico (only available in the US)
• Chromatin Shearing • Chromatin Shearing • DNA Shearing • Cell and tissue disruption • Cell and tissue disruption (e.g. NGS library preparation) Application • RNA Shearing • Chromatin Shearing • Cell and tissue disruption
Standard, basic model with reliable Upgrade of Bioruptor® Standard Provides high-throughput ability Description performance across applications. with timing and temperature and processes up to 12 samples Summary Includes timing control. control. simultaneously with timing and temperature control.
# of tubes # of tubes # of tubes Tube size Tube size Tube size processed processed processed
Troughput and 0.5 ml 12 0.5 ml 12 0.1 ml 12 multiplexing 1.5 ml 6 1.5 ml 6 0.65 ml 12 10 ml 6 10 ml 6 1.5 ml 6 15 ml 6 15 ml 6 15 ml 6 50 ml 3 50 ml 3
Sound-proofing Metal soundproof box Metal soundproof box Not applicable
Includes overheat shutdown Control system for regulated water Control system for regulated water Monitoring protection flow between Bioruptor® and Water flow between Bioruptor® and Water ability and Cooler. Includes overheat shutdown Cooler. control systems protection.
• 0.5 ml Bioruptor® Microtubes • 0.5 ml Bioruptor® Microtubes • 0.1 ml Bioruptor® Microtubes • 1.5 ml TPX Microtubes • 1.5 ml TPX Microtubes • 0.65 ml Bioruptor® Microtubes • 10 ml Tube • 10 ml Tube • 1.5 ml Bioruptor® Microtubes Consumables • 15 ml TPX Tubes • 15 ml TPX Tubes • 15 ml Bioruptor® Tubes • 15 ml Bioruptor® Tubes and sonication beads Innovating Epigenetic Solutions PAGE 14 STEP 3 IMMUNOPRECIPITATION f hPsq niois vial, h lret ubr of number largest antibodies onthemarket for epigenetics. the available, antibodies ChIP-seq of ChIP-seq procedures and results. Browse our latest offering our about information obtain to us Contact metric. control quality antibody an as sequencing performs now Diagenode in interest As epigenetics. trusted performing ChIP on for a genome most – marketwide scale continually the the grows, on providing antibodies to committed is Diagenode market antibodies for theepigenetics Largest numberofvalidated DIAGENODE CHROMATIN IMMUNOPRECIPITATION
antibodies become available. better if even experiments future in "consistency" maintain to simply antibody suboptimal a with assay ChIP every and each optimize must they performance.Oftentimes, antibody with satisfied be necessarily not researchersmay However, products. well-cited and results, comparative antibody, the interest, researchers of often antibody ChIP point their to choose their they how prior about experiencesasked When with antibodies. ChIP of quality the is crucial Particularly before. ever than ChIP of steps all in consistency of and accuracy experimental level higher a requires methods. however, other ChIP-seq, over Successful approach the of accuracy the studies, given chromatin for method standard a as established of sequencing data. ChIP sequencing (ChIP-seq) has now sets been from large of analyses time the to qPCR over to analysis evolved gel PCR have tools research Epigenetic Diagenode’s antibodies? Why shouldyou choose pages to seeifyour favorite target isavailable. Diagenode’sweb program.Visit validationantibody antibody continual our with validation upon sale for antibodies new additional An Among 400 antibodies today, more than 150 are ChIP-grade. chromatin associated proteins, andmodifiedbasesofDNA. quality.currentcollectionThe targets histonemodifications, highest the of continuallyexpertstonovelantibodies deliver for epigenetics research. We assign our R&D and production exclusivelydedicated is antibodies of selection Diagenode's Our dedication Heptromic, Dischrom, andothers. Blueprint, as: such consortiums epigenetic active many in world-class partner key a is Diagenode (BGI). Institute Genomic Beijing with the collaborating or (California) Davis UC as such researchorganizations epigenetic growing community, the in scientific player strong a is Diagenode Involvement intheacademic network problem? How did Diagenode this address quality 80 are ChIP-seq grade ChIP-seq are 80 . We regularly releaseregularly We . PAGE 15
Strict quality standards reagents that have been established as standards for Next- Generation Sequencing by the research community. We use As our partners and clients require comprehensive different sequencing platforms in-house (e.g. Illumina GAII information about an antibody to be available, we indicate and Ion Torrent’s PGM) for our validation projects to provide complete specifications on our technical data sheets. a more flexible range of products and customer support for Only data generated internally or by official partners are a variety of NGS users. considered acceptable. A bioinformatics QC pipeline is set up to 1) constantly control and objectively evaluate every We have the know-how to perform every ChIP-seq step step of the antibody production workflow and 2) facilitate using the same tools as our customers, and we can provide improvements in our processes. expert support from sample preparation to data analysis.
Scale 50 Mb hg18 chrX: 50,000,000 100,000,000 150,000,000 300 -
A380-004 H3K9/14ac Batch1 1 _ 300 -
A381-004 H3K9/14ac Batch2 1 _ 300 -
DA-0010 H3K9/14ac Batch3 1 _ RefSeq Genes
We pay particular attention to the batch-to-batch variations For the latter we have developed a powerful bioinformatics (see above) and thus keep track of every lot in order to make system that includes optimized software tools for the above them comparable. mentioned sequencers, from base calling through reference alignment to peak detection and extended tertiary analysis. Robust and standardized production This tremendous dedication to time and material resources has enabled us to select only the best performing candidates. In our pursuit of achieving the highest quality, Diagenode only uses thoroughly tested and validated methods for antibody development and production. The standardized methods Expert technical support provide uniform conditions, which in turn guarantees high The Diagenode sales and technical teams consist of product quality without technical bias. experts with actual epigenetics bench experience. Our We apply the same quality standards to our bioinformatics. team’s expertise is unmatched as we provide quick and The robust QC pipeline incorporates only well-established, expert advice for the optimization of manual or automated properly tested informatics elements to analyze information epigenetic assays. from all possible sources to evaluate antibodies. The Diagenode team organizes trainings and workshops on a regular basis. If you are interested, please contact us at the Innovation above contact information. Diagenode has developed innovative tools such as DNA and chromatin shearing equipment (Bioruptor®), automated devices (IP-Star® automated workstation), and unique
Please do not hesitate to contact our customer support team if you have any questions about the design of your ChIP-seq experiment or the bioinformatics data analysis. Contact for Europe, Asia, Oceania and Africa: [email protected] ASK THE Contact for North and South America: EXPERTS [email protected]
www.diagenode.com | PAGE 16 DIAGENODE CHROMATIN IMMUNOPRECIPITATION
A safe bet - Diagenode implements the most rigorous antibody validation process on the market to produce ChIP-seq grade antibodies
In many cases, researchers must validate commercially- The role of bioinformatics available antibodies, and they may often find that the To maintain high quality antibody production, Diagenode antibodies do not work. Diagenode, however, exclusively minimizes errors and variability (like lot-to-lot variations) by dedicates antibody research and production for the field of standardizing methods. ChIP-seq grade antibody validation epigenetics, and as thus is a leading antibody provider. Our requires quick and robust analysis of a massive amount superior-quality antibodies are widely used in the epigenetics of data. Our unique scripts and analysis pipeline utilizes community. In addition, Diagenode actively participates in the bioinformatics expertise enabling consistently high-quality BLUEPRINT Consortium, where we have been selected as antibodies. Find the methodology details below. the exclusive antibody supplier. BLUEPRINT a large scale, 30 million euro epigenetic research project spans 41 institutes and companies and aims to decipher the human epigenetic Turning numbers into quality code by sequencing at least100 human epigenome Real expertise is needed to establish a proper ChIP-seq analysis pipeline. There are many checkpoints of quality The importance of validation control (sample quality, sequencing quality, antibody Oftentimes antibody quality is unknown until the results of a quality, reference alignment and peak detection etc.). No project. Antibody suppliers may test for antibody specificity two antibodies behave in the same way – this it is crucial to and sensitivity, usually with dot blots and ELISAs, but few understand the underlying biological mechanisms in order have the complete validation process that Diagenode to choose the correct software tools and settings for each performs. For example, Diagenode tests a number of individual case. In the table below we demonstrate distinct ChIP-seq grade antibodies with both ChIP and ChIP-seq ChIP-seq results of two antibodies against H3K4me3 and experiments in addition to Western blots, Dot blots, ELISAs, H3K27me3 histone marks; both antibodies are working and immunofluorescence. Thus we can guarantee that optimally in the example. our ChIP-seq grade antibodies will work optimally in your Since a universal ‘gold standard’ ChIP-seq QC criteria list experiments – and unlike our competitors, all of our results or QC procedure does not exist, bioinformatics expertise are published in our technical datasheets on our website. is required to analyze the different quality parameters of antibody production. At Diagenode, we have this expertise. Our bioinformaticians can turn numbers and raw data into meaningful biological, chemical, and physiological antibody features, data that puts the power behind our ultra-high quality antibodies.
Innovating Epigenetic Solutions PAGE 17
Comparisons, comparisons, comparisons! Broad Institute datasets generated in the frame of the Encode project. The enrichments gained with our ChIP-seq grade Monitoring the enrichment statistics alone is not enough. antibodies always show a high similarity to this data (often Arguably the best way to check how good a ChIP-seq over 90%). Selecting relevant regions before comparison is a validation experiment is, is to compare the results (the good idea to reduce false peaks. enriched regions) to reference datasets. Since ‘gold standard’ datasets are, again, impossible to find, our best We also use internal controls for comparisons. Identical option is to make as many relevant comparisons as we can results from replicas are always reassuring, but Diagenode and observe the similarity. Compensation for differences in strives further: we also compare with datasets from read numbers, background levels etc. is another challenge different lots, different protocols and different methods, like that requires high-level bioinformatics, as well as obtaining ChIP-seq results from an Illumina GAIIx sequencer and a the proper results (scatter plots, Venn diagrams etc.) and Life Technologies Personal Genome Machine. Therefore their correct interpretation. we can check variation on different levels and minimize any differences, which leads to a consistent and optimal performance of our antibodies, and this is why we can offer the gold standard in ChIP-seq grade antibodies today.
Public datasets are easy to access, but one must be cautious: only similarity to an acknowledged high quality dataset will confirm our good results. Therefore we always use the best accessible data for comparisons of a given antibody, like the
Typical ChIP-seq profiles and their characteristics
H3K3me3 H3K27me3
Type of chromatin enrichment Short, spike-like peaks, even few hundred bases Long enriched regions, even several megabases
Easy to distinguish with the relatively high Hard to distinguish, enrichment is relatively low, Distinction from background enrichment, resulting in high peak significance the peaks’ flanking regions are running into the scores background, significance scores are low
Preferred enrichment Selecting outstanding regions based on Comparing adjacent genome windows after detection method (example mathematical modeling of read distributions windowed tag counting, selecting and merging software tool) (MACS) the significant ones (Sicer)
Genome coverage of Low (even less than 1/10th of the whole genome) High (even more than 1/3rd of the whole genome) enrichments
Required read number for 2 to 5 million 35 bp reads can be enough for good 20 to 30 million 35 bp reads can be inadequate for adequate coverage saturation proper saturation
Reads in peaks ratio Can be low (even 1-10%) Can be high (60-80%)
Association with biological High promoter affinity (usually> 70% of peaks in Moderate coding region affinity (usually >50% of features HeLa cells) peaks in HeLa cells)
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Antibody rating
Premium 5 star rating in our catalog. Fully validated antibodies, selected for high specificity and sensitivity. These antibodies provide superior performance for any application with a main focus on ChIP-seq.
Classic 4 star rating in our catalog. Validated through standard procedures in the market for most immunoassays including ChIP.
Pioneer 3 star rating in our catalog. Novel antibodies successfully validated for specify against corresponding antigen available at low cost.
Check out our sample sizes
PREMIUM Cat. No. Format Applications Purity
C15200081-10 10 µg
5-mC monoclonal antibody 33D3 C15200081-100 100 µg MeDIP-seq, ELISA, Dot blot, IF, FISH, Southern blot Affinity purified
C15200081-500 500 µg
C15410194-10 10 μg H3K4me1 polyclonal antibody ChIP/ChIP-seq, ELISA, Dot blot, WB, IF Affinity purified C15410194-50 50 μg
C15410003-10 10 μg H3K4me3 polyclonal antibody ChIP/ChIP-seq, ELISA, Dot blot, WB, IF Affinity purified C15410003-50 50 μg
C15410193-10 10 μg H3K9me3 polyclonal antibody ChIP/ChIP-seq, ELISA, Dot blot, WB, IF Affinity purified C15410193-50 50 μg
C15410200-10 10 μg ChIP/ChIP-seq, ELISA, Dot blotting/Peptide array, Affinity purified H3K9/14ac polyclonal antibody WB, IF C15410200-50 50 μg
C15410196-10 10 μg H3K27ac polyclonal antibody ChIP/ChIP-seq, ELISA, Dot blot, WB, IF Affinity purified C15410196-50 50 μg
C15410195-10 10 μg H3K27me3 polyclonal antibody ChIP/ChIP-seq, ELISA, Dot blot, WB, IF Affinity purified C15410195-50 50 μg
C15410192-10 10 μg H3K36me3 polyclonal antibody ChIP/ChIP-seq, ELISA, Dot blot, WB, IF Affinity purified C15410192-50 50 μg
C15410207-10 10 μg ChIP/ChIP-seq, ELISA, Dot blotting/Peptide array, WB, Affinity purified H4K20me3 polyclonal antibody IF C15410207-50 50 μg
C15410201-10 10 μg H2A.Z polyclonal antibody ChIP/ChIP-seq, ELISA, WB, IF Affinity purified C15410201-50 50 μg
C15410202-10 10 μg ChIP/ChIP-seq, ELISA, Dot blotting/Peptide array, Affinity purified H2A.Zac polyclonal antibody WB, IF C15410202-50 50 μg
CRISPR/CAS9 Cat. No. Format Applications Purity ANTIBODIES
C15200230-10 10 µg S. aureus CRISPR/Cas9 monoclonal IF, IP, WB Protein G purified antibody C15200230 50 µg
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C15310260-20 20 µl S. aureus CRISPR/Cas9 polyclonal IF, IP, WB Whole antiserum antibody (N-terminal) C15310260 100 µl
C15310259-20 20 µl S. aureus CRISPR/Cas9 polyclonal IF, IP, WB Protein G purified antibody (C-terminal) C15310259 100 µl
C15200223-14 14 µg CRISPR/Cas9 C-terminal IF, IP, WB Protein G purified monoclonal antibody C15200223-50 50 µg
CRISPR/Cas9 monoclonal antibody C15200229 50 μg/25 μl ChIP-qPCR (ab), WB, IP, IF Protein G purified
C15200216-10 10 µg CRISPR/Cas9 monoclonal antibody WB, IF Protein G purified 4G10 C15200216-50 50 µg
C15310258-20 20 μl CRISPR/Cas9 polyclonal antibody ChIP, IF, IP, WB Whole antiserum C15310258 100 μl
CRISPR/Cas9 - HRP monoclonal C15200215 500 µl WB Protein G purified antibody 7A9
CRISPR/Cas9 monoclonal antibody C15200203 50 µg IF, IP, WB Protein G purified 7A9
CLASSIC Cat. No. Format Applications Purity
C15220001-20 20 µg hMeDIP, IF, Dot blot, IHC Protein A/G purified
5-hmC monoclonal antibody (rat) C15220001-50 50 µg hMeDIP, IF, Dot blot Protein A/G purified
C15220001-100 100 µg hMeDIP, IF, Dot blot Protein A/G purified
C15310210-20 20 µl hMeDIP, Dot blot, ELISA Whole serum 5-hmC polyclonal antibody (rabbit) C15310210-100 100 µl hMeDIP, Dot blot, ELISA Whole serum
5-hmC polyclonal antibody C15410205-50 50 μg hMeDIP, ELISA, Dot blot Affinity purified
C15200006-100 100 µg MeDIP, IF Affinity purified 5-mC monoclonal antibody cl. b C15200006-500 500 µg MeDIP, IF Affinity purified
5-formylcytosine polyclonal antibody C15310200 100 µl DNA IP, ELISA Whole serum
5-mC monoclonal antibody for ICC/ C15200003 50 μg MeDIP, Dot blot, IF, FISH Protein A purified IF 6X His epitope tag monoclonal C15200208 100 µg ELISA, WB, Immunoprecipitation Affinity purified antibody
C15200167-10 10 μg Ago (Argonautes) monoclonal C15200167-050 50 μg RIP, HITS CLIP, IHC, IF, WB Protein A purified antibody C15200167-100 100 μg
AML1-ETO polyclonal antibody C15310197 100 μl ChIP/ChIP-seq, ELISA, WB Whole antiserum
ASH1L polyclonal antibody C15410304 100 µg ELISA, IHC, IF Affinity purified
BCL6 polyclonal antibody C15410221 25 µg, 100 µg ChIP, IHC, Immunoprecipitation, WB Affinity purified
BRN2 polyclonal antibody C15410223 25 µg, 100 µg ChIP, CC/IF, IHC, Immunoprecipitation, WB Affinity purified
C/EBP alpha polyclonal antibody C15410225 25 µg, 100 µg ChIP, Immunoprecipitation, WB Affinity purified
CBFb polyclonal antibody C15310002 100 μl ChIP/ChIP-seq, ELISA Whole antiserum
CBP polyclonal antibody C15410224 25 µl, 100 µl ChIP, ICC/IF, IHC, Immunoprecipitation, WB Affinity purified
Cdc73 polyclonal antibody C15310019 100 μl ChIP, WB, IP Whole serum
Cdc73 polyclonal antibody C15410019 50 μg ChIP, WB, IF Affinity purified
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CLASSIC Cat. No. Format Applications Purity
CIITA polyclonal antibody C15410062 50 μg ChIP Affinity purified
C15410210-10 10 μg CTCF polyclonal antibody ChIP/ChIP-seq, ELISA, WB Affinity purified C15410210-50 50 μg
CTNNB1 polyclonal antibody C15410222 25 µg, 100 µg ChIP, ICC/IF, IHC, Immunoprecipitation, WB Affinity purified
CXXC1 polyclonal antibody C15410315 50 μg ChIP/ChIP-seq, WB, IF Affinity purified
DNMT1 polyclonal antibody C15410227 25 µl, 100 µl ChIP, ICC/IF, IHC, Immunoprecipitation, WB Affinity purified
DNMT3A polyclonal antibody C15410084 50 µg ELISA, WB, IP Affinity purified
DNMT3A polyclonal antibody C15410085 50 µg ELISA, WB, IP Affinity purified
DNMT3A polyclonal antibody C15410086 50 µg ELISA, WB, IP Affinity purified
DP1 polyclonal antibody C15410228 25 µg, 100 µg Immunoprecipitation, WB Affinity purified
E2F6 polyclonal antibody C15410314 50 µg ChIP/ChIP-seq, WB, IF Affinity purified
EGFP polyclonal antibody C15410074 50 µg IF Affinity purified
ER alpha monoclonal antibody C15100066 100 μl ChIP/ChIP-seq, ELISA, WB, GSA, IC, IP Ascites fluid
C15200009-10 10 μg ER alpha monoclonal antibody ChIP, WB, IC Protein A/G purified C15200009-50 50 μg
ERR alpha polyclonal antibody C15410229 25 µl, 100 µl ChIP, Immunoprecipitation, WB Affinity purified
ERR alpha polyclonal antibody C15410230 25 µl, 100 µl ChIP, Immunoprecipitation, WB Affinity purified
ETO polyclonal antibody C15310001 100 μl ChIP/ChIP-seq, ELISA Whole antiserum
EZH2 monoclonal antibody C15200180 50 μg ChIP Protein A/G purified
EZH2 polyclonal antibody C15410039 50 μg ChIP/ChIP-seq, WB, IC Affinity purified
FLAG monoclonal antibody C15200209 100 µg ELISA, WB Affinity purified
FOXA1 polyclonal antibody C15410231 25 µg, 100 µg ChIP, ICC/IF, IHC, Immunoprecipitation, WB Affinity purified
FOXM1 polyclonal antibody C15410232 25 µg, 100 µg IHC, WB Affinity purified
FUBP1 polyclonal antibody C15410233 25 µg, 100 µg ChIP, ICC/IF, IHC, Immunoprecipitation, WB Affinity purified
GR monoclonal antibody C15200010 50 μg ChIP/ChIP-seq, ELISA, WB, GSA, IC, Flow Cyt, IP Protein A/G purified
GST monoclonal antibody C15200207 500 µg ELISA, WB Protein A purified
GST polyclonal antibody C15430001 100 µg ELISA, WB IgG fraction
GST polyclonal antibody C15410268 100 µg ELISA, WB, Immunohistochemistry Affinity purified
C15410173-10 10 µl H2A.Zac polyclonal antibody ChIP/ChIP-seq, ELISA, Dot blot, WB, IF Affinity purified C15410173 50 μg
H2AK119ub polyclonal antibody C15410002 50 μg ChIP, ELISA, Dot blot, WB Affinity purified
C15410215-10 10 µl H2AK5ac polyclonal antibody ChIP/ChIP-seq, ELISA, Dot blot, WB Affinity purified C15410215 50 μg
H2Apan polyclonal antibody C15410166 50 μg ChIP, ELISA, Dot blot Affinity purified
H2A.XS139p polyclonal antibody C15410219 50 μg ELISA, Dot blot, WB Whole serum
C15400212-10 10 µl H2BK12ac polyclonal antibody ChIP/ChIP-seq, ELISA, Dot blot, WB Affinity purified C15410212 50 μg
Innovating Epigenetic Solutions PAGE 21
CLASSIC Cat. No. Format Applications Purity
C15410220-10 10 µl H2BK15ac polyclonal antibody ChIP/ChIP-seq, ELISA, Dot blot, WB Affinity purified C15410220 50 μg
H2Bpan polyclonal antibody C15410157 50 μg ChIP, ELISA, Dot blot Affinity purified
H3 Pan polyclonal antibody C15310135 100 μl ChIP, ELISA, Dot blot, WB Whole serum
C15410310-10 10 µg H3K14ac polyclonal antibody ChIP - ELISA - Dotblot - Wb Affinity purified C15410310 50 µg
C15410139-10 10 µl H3K18ac polyclonal antibody ChIP/ChIP-seq, ELISA, Dot blot, WB, IF Affinity purified C15410139 50 μg
H3K18ac polyclonal antibody C15310139 100 μl ELISA, Dot blot, WB Whole antiserum
H3K18me1 polyclonal antibody C15410290 50 µg ChIP, IF, WB, Immunochemistry, Dot blot Affinity purified
H3K18me2 polyclonal antibody C15410291 50 µg ChIP, IF, WB, Immunochemistry, Dot blot Affinity purified
H3K18me3 polyclonal antibody C15410292 50 µg ChIP, IF, WB, Immunochemistry, Dot blot Affinity purified
H3K23ac polyclonal antibody C15310140 100 μl ELISA, Dot blot, WB Whole antiserum
H3K27ac monoclonal antibody C15200184 50 μg ChIP, ELISA, WB, IF Protein A/G purified
C15410174-10 10 µl H3K27ac polyclonal antibody ChIP/ChIP-seq, ELISA, Dot blot, WB, IF Affinity purified C15410174 50 μg
H3K27me1 polyclonal antibody C15410045 50 μg ChIP, ELISA, Dot blot, WB, IF Affinity purified
H3K27me2 polyclonal antibody C15410046 50 μg ChIP, ELISA, Dot blot, WB Affinity purified
H3K27me2/3 monoclonal antibody C15200014 50 μg ChIP, Dot blot, WB, IF Protein A/G purified
H3K27me3 monoclonal antibody C15200181 50 μg ChIP, ELISA, WB, IF Protein A/G purified
H3K27me3 polyclonal antibody C15310069 100 µl ChIP, ELISA, Dot blot, WB, IF Whole serum
H3K27me3 polyclonal antibody C15410069 50 μg ChIP/ChIP-seq, ELISA, Dot blot, WB, IF Affinity purified
H3K27me3S28p polyclonal antibody C15310091 100 μl ChIP/ChIP-seq, ELISA, Dot blot, WB, IF, IP Whole serum
H3K27me3S28p polyclonal antibody C15410091 50 μg ChIP/ChIP-seq, ELISA, Dot blot, WB Affinity purified
H3K27me3S28p polyclonal antibody C15410293 50 µg ChIP, IF, WB, Immunochemistry, Dot blot Affinity purified
C15410307-10 10 µg H3K36ac polyclonal antibody ChIP/ChIP-seq - ELISA - Dot blotting - WB Affinity purified C15410307 50 μg
H3K36me1 polyclonal antibody C15410089 50 μg ChIP, ELISA, Dot blot, WB Affinity purified
H3K36me2 monoclonal antibody C15200182 50 μg ChIP, ELISA, Dot blot, WB Protein A/G purified
H3K36me2 polyclonal antibody C15310127 100 μl ChIP/ChIP-seq, ELISA, Dot blot, WB, IF Whole serum
H3K36me3 monoclonal antibody C15200183 50 μg ChIP, ELISA, Dot blot, WB, IF Protein A/G purified
H3K36me3 polyclonal antibody C15310058 100 μl ChIP/ChIP-seq, ELISA, Dot blot, WB Whole serum
C15410058-10 10 μl H3K36me3 polyclonal antibody ChIP/ChIP-seq, ELISA, Dot blot, WB Affinity purified C15410058-50 50 μg
H3K37me1 polyclonal antibody C15410295 50 µg WB, Dot blot Affinity purified
H3K4ac polyclonal antibody C15410165 50 μg ChIP, ELISA, Dot blot, WB, IF Affinity purified
H3K4ac polyclonal antibody C15410322 50 μg ChIP/ChIP-seq, ELISA, Dot blot, WB, IF Affinity purified
H3K4me1 monoclonal antibody C15200150 50 μg ChIP, ELISA, WB, IF Protein A/G purified
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CLASSIC Cat. No. Format Applications Purity
H3K4me1 polyclonal antibody C15310037 100 μl ChIP, ELISA, Dot blot, WB Whole serum
H3K4me1 polyclonal antibody C15410037 50 μg ChIP/ChIP-seq, ELISA, Dot blot, WB Affinity purified
H3K4me1T6p polyclonal antibody C15410280 50 µg ChIP, Immunohistochemistry, IF, WB, Dot blot Affinity purified
H3K4me2 monoclonal antibody C15200151 50 μg ChIP, ELISA, WB, IF Protein A/G purified
H3K4me2 polyclonal antibody C15310035 100 μl ChIP, ELISA, Dot blot, WB, IF Whole serum
C15410035-10 10 μg H3K4me2 polyclonal antibody ChIP/ChIP-seq, ELISA, Dot blot, WB, IF Affinity purified C15410035-50 50 μg
H3K4me3 monoclonal antibody C15200152 50 μg ChIP/ChIP-seq, ELISA, WB, IF Protein A/G purified
H3K4me3 polyclonal antibody C15310003 100 µl ChIP, ELISA, Dot blot, WB Whole antiserum
H3K4me3 polyclonal antibody C15310030 100 µl Dot blot, WB Whole serum
H3K4me3 polyclonal antibody C15410030 50 μg ChIP/ChIP-seq, Dot blot, WB, IF Affinity purified
H3K4me3 polyclonal antibody C15410203 50 µl ChIP, ELISA, Dot blot, WB Affinity purified
H3K4me3T6p polyclonal antibody C15410281 50 µg ChIP, Immunohistochemistry, IF, WB, Dot blot Affinity purified
H3K4un monoclonal antibody C15200149 50 μg ChIP, ELISA, WB, IF Protein A/G purified
C15410213-10 10 µg H3K56ac polyclonal antibody ChIP/ChIP-seq, ELISA, WB Affinity purified C15410213 50 μg
H3K56me1 polyclonal antibody C15410296 50 µg ChIP, IF, WB, Dot blot Affinity purified
H3K56me3 polyclonal antibody C15410297 50 µg ChIP, IF, WB, Dot blot Affinity purified
H3K64me3 polyclonal antibody C15410211 50 μg ChIP, ELISA, WB Affinity purified
H3K79me1 polyclonal antibody C15310082 100 μl ChIP, ELISA, Dot blot, WB Whole serum
H3K79me1 polyclonal antibody C15410082 50 μg ChIP/ChIP-seq, ELISA, Dot blot, WB Affinity purified
H3K79me2 polyclonal antibody C15310051 100 μl ChIP, ELISA, Dot blot, WB Whole serum
H3K79me2 polyclonal antibody C15410051 50 μg ChIP/ChIP-seq, ELISA, Dot blot, WB Affinity purified
H3K79me3 polyclonal antibody C15310068 100 μl ELISA, Dot blot, WB, IF Whole serum
C15410068-10 10 μg H3K79me3 polyclonal antibody ChIP/ChIP-seq, ELISA, Dot blot, WB Affinity purified C15410068-50 50 μg
C15410005-10 10 μg H3K9/14ac polyclonal antibody ChIP/ChIP-seq, ELISA, Dot blot, WB, IF Affinity purified C15410005-50 50 µg
H3K9ac monoclonal antibody C15200185 50 μg ChIP, ELISA, WB, IF Protein A/G purified
C15410004-10 10 μg H3K9ac polyclonal antibody ChIP/ChIP-seq, ELISA, Dot blot Affinity purified C15410004 50 µg
H3K9acS10p polyclonal antibody C15310102 100 μl ChIP, ELISA, Dot blot, WB, IF Whole serum
H3K9me1 polyclonal antibody C15310065 100 μl ChIP, ELISA, Dot blot, WB, IF Whole serum
H3K9me1 polyclonal antibody C15410065 50 µg ChIP, ELISA, Dot blot, WB Affinity purified
H3K9me2 polyclonal antibody C15410060 50 μg ChIP, ELISA, Dot blot, WB, IF Affinity purified
H3K9me2 monoclonal antibody C15200154 50 μg ChIP, ELISA, WB, IF Protein A/G purified
C15200146-10 10 μg H3K9me3 monoclonal antibody ChIP, Dot blot, WB Affinity purified C15200146-50 50 μg
Innovating Epigenetic Solutions PAGE 23
CLASSIC Cat. No. Format Applications Purity
H3K9me3 monoclonal antibody C15200153 50 μg ChIP, ELISA, WB, IF Protein A/G purified
H3K9me3 polyclonal antibody C15310013 100 μl ChIP, WB Whole antiserum
H3K9me3 polyclonal antibody C15310056 100 μl ChIP, ELISA, Dot blot, WB, IF Whole serum
H3K9me3 polyclonal antibody C15410013 50 µg ChIP, WB Protein G purified
C15410056-10 10 µg H3K9me3 polyclonal antibody ChIP/ChIP-seq, ELISA, Dot blot, WB, IF Affinity purified C15410056 50 μg
Concentrated H3K9me3 monoclonal antibody C15100146 100 µl ChIP, Dot blot, WB supernatant H3K9me3 recombinant antibody C15500003 50 µg ChIP, IF Affinity purified and negative control
H3K9me3S10p polyclonal antibody C15310128 100 μl ChIP, ELISA, Dot blot, WB, IF Whole serum
H3K9un monoclonal antibody C15200187 50 μg ChIP, WB, IF Protein A purified
H3pan monoclonal antibody C15200011 50 μg ChIP, WB Protein A purified
H3R2me2 (asym) polyclonal antibody C15410316 50 μg ChIP, WB Affinity purified
H3R17me2(asym) polyclonal C15310092 100 μl ChIP, ELISA, Dot blot Whole serum antibody
H3R17me2(asym) polyclonal antibody C15410289 50 µg ChIP, IF, WB Affinity purified
H3R17me2(asym)K18ac polyclonal C15410171 50 μg ChIP/ChIP-seq, ELISA, Dot blot, WB, IF Affinity purified antibody
H3R2me1 polyclonal antibody C15410276 50 µg WB Affinity purified
H3R2me2(sym)K4me2 polyclonal ChIP, IF Microscopy, WB, Immunochemistry, Dot C15410294 50 µg Affinity purified antibody blot H3R2me2(sym)T3p polyclonal C15410303 50 µg IF, WB, Dot blot Affinity purified antibody
H3R8me2(asym) polyclonal antibody C15410286 50 µg ChIP, Immunohistochemistry, IF, WB Affinity purified
H3R8me2(sym) polyclonal antibody C15410287 50 µg ChIP, WB Affinity purified
H3S10p polyclonal antibody C15310116 100 μl ChIP, ELISA, Dot blot, WB, IF, IP Whole serum
C15410116-10 10 μg H3S10p polyclonal antibody ChIP, ELISA, Dot blot, WB, IF Affinity purified C15410116-50 50 μg
H3S10pT11p polyclonal antibody C15410288 50 µg ChIP, Immunohistochemistry, IF, WB Affinity purified
H3T3pK4ac polyclonal antibody C15410279 50 µg ChIP, Immunohistochemistry, IF, WB, Dot blot Affinity purified
H3T3pK4me1 polyclonal antibody C15410277 50 µg Immunohistochemistry, IF, WD, Dot blot Affinity purified
H3T3pK4me2 polyclonal antibody C15410278 50 µg ChIP, Immunohistochemistry, IF, WB, Dot blot Affinity purified
H3T6p polyclonal antibody C15410282 50 µg ChIP, Immunohistochemistry, IF, WB, Dot blot Affinity purified
H3T6pK9me1 polyclonal antibody C15410283 50 µg ChIP, Immunohistochemistry, IF, WB, Dot blot Affinity purified
H3T6pK9me2 polyclonal antibody C15410284 50 µg ChIP, Immunohistochemistry, IF Microscopy, WB Affinity purified
H3T6pK9me3 polyclonal antibody C15410285 50 µg ChIP, Immunohistochemistry, IF, WB, Dot blot Affinity purified
Concentrated H4K20me1 monoclonal antibody C15100147 100 µl ChIP, Dot blot, WB supernatant
C15200147-10 10 µl H4K20me1 monoclonal antibody ChIP/ChIP-seq, Dot blot, WB, IF Protein A/G purified C15200147-50 50 μg
H4K20me1 polyclonal antibody C15410034 50 μg ChIP, ELISA, Dot blot, WB Affinity purified
H4K20me1 polyclonal antibody C15310034 100 μl ChIP, ELISA, Dot blot, WB Whole serum
H4K20me2 monoclonal antibody C15200205 100 µg ChIP, ICC/IF, WB Affinity purified
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CLASSIC Cat. No. Format Applications Purity
H4K20me3 polyclonal antibody C15310057 100 μl ChIP, ELISA, Dot blot, WB Whole serum
C15410057-10 10 μl H4K20me3 polyclonal antibody ChIP/ChIP-seq, ELISA, Dot blot, WB, IF Affinity purified C15410057-50 50 μg
Concentrated H4K20me3 monoclonal antibody C15100148 100 μl ChIP, WB, IF supernatant
C15410025-10 10 µl H4K5ac polyclonal antibody ChIP/ChIP-seq, ELISA, Dot blot, WB Affinity purified C15410025 50 μg
C15410021-10 10 μl H4K5,8,12ac polyclonal antibody ChIP/ChIP-seq, ELISA, Dot blot, WB, IF Affinity purified C15410021 50 μg
C15410024-10 10 μl H4K5,8,12,16ac polyclonal antibody ChIP/ChIP-seq, ELISA, Dot blot, IF Affinity purified C15410024 50 μg
H4K8ac polyclonal antibody C15310103 100 μl ChIP, ELISA, Dot blot, WB Whole serum
C15410103-10 10 µl H4K8ac polyclonal antibody ChIP, ELISA, Dot blot, WB, IF Affinity purified C15410103 50 µg
ChIP, Dot blot, ICC/IF, IHC, Immunoprecipitation, H4K8ac polyclonal antibody C15410235 25 µg, 100 µg Affinity purified WB
C15200217-10 10 µl H4K8ac monoclonal antibody ChIP - Dot blot - WB Protein A purified C15200217 50 µg
H4K12ac polyclonal antibody C15410302 50 µg IF, Immunohistochemistry, WB, Dot Blot Affinity purified
C15200218-10 10 µl H4K12ac monoclonal antibody ChIP - Dot blot - WB Protein A purified C15200218 50 µg
C15200219-10 10 µl H4K16ac monoclonal antibody ChIP - Dot blot - WB Protein A purified C15200219 50 µg
H4K16ac polyclonal antibody C15410300 50 µg ChIP, IF, WB, Immunochemistry, Dot Blot Affinity purified
H4K20me2 polyclonal antibody C15410301 50 µg ChIP, IF, WB, Immunochemistry Affinity purified
H4pan polyclonal antibody C15410156 50 µg ChIP, ELISA, Dot blot Affinity purified
H4S1p polyclonal antibody C15410298 50 µg ChIP, IF, WB, Immunohistochemistry, Dot Blot Affinity purified
H4R3me1 polyclonal antibody C15410299 50 µg ChIP, IF, WB, Immunochemistry, Dot Blot Affinity purified
HA tag monoclonal antibody C15200190 50 μg WB, IS Protein A purified
HDAC1 monoclonal antibody C15200144 50 μg ChIP, WB, IF Protein A/G purified
HDAC1 polyclonal antibody C15410053 50 μg ChIP/ChIP-seq, ELISA, WB, IF Affinity purified
Concentrated HDAC1 monoclonal antibody C15100144 100 μl ChIP, WB supernatant
HDAC2 monoclonal antibody C15200201 50 μg ChIP/ChIP-seq, WB, IF Protein A purified
HDAC3 monoclonal antibody C15200145 50 μg ChIP, IF Protein A/G purified
Concentrated HDAC3 monoclonal antibody C15100145 100 µl ChIP supernatant
HDAC5 polyclonal antibody C15410275 100 µg ELISA, WB, Immunohistochemistry, IF Microscopy Affinity purified
HIF1 alpha polyclonal antibody C15410234 25 µg, 100 µg ChIP, ICC/IF, IHC, Immunoprecipitation, WB Affinity purified
His tag monoclonal antibody C15200191 50 μg WB, IF Protein A purified
HJURP monoclonal antibody C15200228 50 µg WB, IF Protein A/G purified
hnRNP A/B polyclonal antibody C15410263 25 µg, 100 µg ICC/IF, IHC, WB Affinity purified
Innovating Epigenetic Solutions PAGE 25
CLASSIC Cat. No. Format Applications Purity
HP1α, ß and γ polyclonal antibody C15410071 50 µg ChIP, WB, IF Affinity purified
Huntingtin monoclonal antibody C15200226 50 μg WB, IF Protein A/G purified
JMJD6 polyclonal antibody C15410318 50 µg ChIP/ChIP-seq, WB, IHC Affinity purified
ChIP/ChIP-seq, ChIP, ICC/IF, IHC, KAP1 polyclonal antibody C15410236 25 µg, 100 µg Affinity purified Immunoprecipitation, WB
KDM6A polyclonal antibod C15410237 25 µg, 100 µg ChIP, ICC/IF, IHC, Immunoprecipitation, WB Affinity purified
L3MBTL1 polyclonal antibody C15410023 50 µg WB, IP Protein G purified
L3MBTL2 polyclonal antibody C15410238 25 µg, 100 µg ChIP, Immunoprecipitation, WB Affinity purified
LDB1 polyclonal antibody C15410269 100 µg ELISA, WB Affinity purified
LDB2 polyclonal antibody C15410271 100 µg Immunohistochemistry, WB Affinity purified
LITAF polyclonal antibody C15410265 25 µg, 100 µg IHC, WB Affinity purified
LSD1 polyclonal antibody C15410067 50 µg ChIP/ChIP-seq, ELISA, WB, IF Affinity purified
LSD1 polyclonal antibody C15410028 50 µg ChIP, WB Affinity purified
LSD1 polyclonal antibody C15410125 50 µg ELISA, WB, IP Affinity purified
MBD1 polyclonal antibody C15410078 50 μg ChIP, ELISA, WB Affinity purified
ChIP, ICC/IF, Immunohistochemistry, MCM6 polyclonal antibody C15410239 25 µg, 100 µg Affinity purified Immunoprecipitation, Western blot
MECP2 monoclonal antibody C15200225 50 μg WB, IF Protein A/G purified
C15410052-10 10 μg MeCP2 polyclonal antibody ChIP, ELISA, WB Affinity purified C15410052-50 50 μg mTOR polyclonal antibody C15410262 25 µg, 100 µg ChIP, ICC/IF, IHC, Immunoprecipitation, WB Affinity purified
MYH11 polyclonal antibody C15310254 100 μl ChIP/ChIP-seq, ELISA, WB Whole antiserum
NF-E2 polyclonal antibody C15410240 25 µg, 100 µg ChIP/ChIP-seq, ChIP, ICC/IF, WB Affinity purified
NFKB cRel polyclonal antibody C15310257 100 µl ELISA, Gel Shift, WB Whole antiserum
NFKB p50 polyclonal antibody C15310255 100 µl ELISA, WB Whole antiserum
ChIP/ChIP-seq , ELISA, Gel Shift, NFKB p65 polyclonal antibody C15310256 100 µl Whole antiserum Immunohistochemistry, WB ChIP/ChIP-seq, ChIP, ICC/IF, Immunoprecipitation, NFYB polyclonal antibody C15410241 25 µg, 100 µg Affinity purified WB
NRF1 monoclonal antibody C15200013 50 μg ChIP/ChIP-seq, WB Protein A purified
NRF2 polyclonal antibody C15410242 25 µg, 100 µg ChIP, ICC/IF, IHC, WB Affinity purified
OCT4 polyclonal antibody C15410305 50 μg ChIP/ChIP-seq, ELISA, WB Affinity purified
ORC2 monoclonal antibody C15200198 100 µl WB Whole antiserum p53 polyclonal antibody C15410083 50 μg ChIP/ChIP-seq, ELISA, WB Affinity purified p53K372me1 polyclonal antibody C15410048 50 µg ChIP, WB Protein G purified
PAD4 polyclonal antibody C15410244 25 µl, 100 µl ChIP, ICC/IF, IHC, WB Affinity purified
Paf1 polyclonal antibody C15310016 100 µl WB, IF, IP Whole antiserum
Paf1 polyclonal antibody C15410015 50 μg ChIP, WB Affinity purified
Paf1 polyclonal antibody C15410016 50 µg WB, IF, IP Protein G purified
PARP1 polyclonal antibody C15410245 25 µg, 100 µg ChIP, ICC/IF, IHC, Immunoprecipitation, WB Affinity purified
PAX8 polyclonal antibody C15410246 25 µg, 100 µg ChIP, ICC/IF, IHC, IHC, Immunoprecipitation, WB Affinity purified
www.diagenode.com | PAGE 26 DIAGENODE CHROMATIN IMMUNOPRECIPITATION
CLASSIC Cat. No. Format Applications Purity
PKNOX1 polyclonal antibody C15410267 25 µg, 100 µg IF, Immunoprecipitation, WB Affinity purified
Pol II monoclonal antibody C15100055 100 μl ChIP/ChIP-seq Ascites fluid
C15200004-10 10 μg Pol II monoclonal antibody ChIP/ChIP-seq, ELISA, WB, IF Protein A purified C15200004-50 50 μg
Pol II S2p monoclonal antibody C15200005 50 μg ChIP/ChIP-seq, ELISA, WB, IF Protein A purified
Pol II S5p monoclonal antibody C15200007 50 μg ChIP/ChIP-seq, ELISA, WB, IF Protein A purified
PPARG polyclonal antibody C15410133 50 μg ChIP, ELISA, WB Affinity purified
PRMT7 polyclonal antibody C15410247 25 µg, 100 µg ChIP, ICC/IF, IHC, Immunoprecipitation, WB Affinity purified
C15310155-10 10 µl
RARA polyclonal antibody C15310155-20 20 μl ChIP/ChIP-seq, ELISA, WB Whole serum
C15310155-100 100 μl
Protein G Affinity RbAp48 monoclonal antibody C15200206 100 µg ChIP, ICC/IF, Immunoprecipitation, WB Purified
RFX-AP polyclonal antibody C15410061 50 μg ChIP Affinity purified
RING1B polyclonal antibody C15430002 100 µg ELISA, Immunofluorescence Microscopy, WB Affinity purified
RNF2 polyclonal antibody C15410313 50 µg ChIP/ChIP-seq, WB, IF Affinity purified
ROR beta polyclonal antibody C15410001 100 μl WB, GSA, IF Affinity purified
Rsf1 monoclonal antibody C15100041 100 µl WB, IF, IP Ascites fluid
Rsf1 monoclonal antibody C15200041 50 µg WB, IF, IP Protein G purified
Rtf1 polyclonal antibody C15310018 100 µl ChIP, WB, IF, IP Whole antiserum
Rtf1 polyclonal antibody C15410018 50 µg ChIP, WB, IF, IP Protein G purified
SATB1 polyclonal antibody C15410248 25 µl, 100 µl ChIP, IHC, Immunoprecipitation, WB Affinity purified
SETDB1 polyclonal antibody C15410249 25 µl, 100 µl ChIP, Immunoprecipitation, WB Affinity purified
ChIP/ChIP-seq, ChIP, ICC/IF, IHC, SIN3A polyclonal antibody C15410250 25 µg, 100 µg Affinity purified Immunoprecipitation, WB
Ski3 polyclonal antibody C15410011 150 µg WB, IF, IP Protein G purified
Ski8 polyclonal antibody C15410012 150 µg ChIP, WB Protein G purified
ChIP/ChIP-seq, ChIP, ELISA, WB, SMAD1 polyclonal antibody C15410274 100 µg Affinity purified Immunoprecipitation
SMAD2 polyclonal antibody C15410251 25 µg, 100 µg ChIP, ICC/IF, Immunoprecipitation, WB Affinity purified
SMAD2 polyclonal antibody C15410273 100 µg ELISA, WB Affinity purified
SMAD3 polyclonal antibody C15410252 25 µl, 100 µl ChIP, ICC/IF, IHC, Immunoprecipitation, WB Affinity purified
SMARCB1 polyclonal antibody C15410317 50 µg ChIP, WB Affinity purified
ChIP, ICC/IF, IHC (Formalin-fixed paraffin- SMYD3 polyclonal antibody C15410253 25 µg, 100 µg Affinity purified embedded sections), Immunoprecipitation, WB
Snf2h monoclonal antibody C15100040 100 µl WB, IF, IP Ascites fluid
Snf2h monoclonal antibody C15200040 50 µg WB, IF, IP Protein G purified
STAT2 monoclonal antibody C15200210 100 µg ELISA, WB, Immunohistochemistry Protein A purified
STAT6 polyclonal antibody C15410254 25 µg, 100 µg ChIP, IHC, Immunoprecipitation, WB Affinity purified
Suz12 polyclonal antibody C15310029 100 μl ChIP, WB Whole serum
Suz12 polyclonal antibody C15410029 50 μg ChIP, WB Affinity purified
TAL1 monoclonal antibody C15200012 50 μg ChIP/ChIP-seq Affinity purified
Innovating Epigenetic Solutions PAGE 27
CLASSIC Cat. No. Format Applications Purity
TAF1 polyclonal antibody C15410272 100 µg ELISA, WB, Immunochemistry Affinity purified
ChIP/ChIP-seq, ChIP, IF, IHC, Immunoprecipitation, TARDBP polyclonal antibody C15410266 25 µg, 100 µg Affinity purified WB
TBP monoclonal antibody C15200002 100 µg ChIP/ChIP-seq, WB Protein A/G purified
TDG monoclonal antibody C15200227 50 μg WB, IF Protein A/G purified
TET2 monoclonal antibody C15200179 50 μg WB, IP Protein G purified
TET2 polyclonal antibody C15410255 25 µl, 100 µl ChIP, ICC/IF, Immunoprecipitation, WB Affinity purified
TFIIE beta polyclonal antibody C15410264 25 µg, 100 µg ChIP, Immunoprecipitation, WB Affinity purified
TFIIF polyclonal antibody C15410256 25 µg, 100 µg ChIP, IP, WB Affinity purified
THOC1 polyclonal antibody C15410243 25 µg, 100 µg ChIP, ICC/IF, IHC, Immunoprecipitation, WB Affinity purified
TIP5 polyclonal antibody C15310090 100 μl ChIP, WB Whole serum
TRRAP polyclonal antibody C15410257 25 µg, 100 µg ChIP, ICC/IF, Immunoprecipitation, WB Affinity purified
TY1-tag monoclonal antibody C15200054 50 μg ChIP, WB IgG fraction
UHRF1 polyclonal antibody C15410258 25 µg, 100 µg ChIP, ICC/IF, IHC, Immunoprecipitation, WB Affinity purified
V5 Epitope tag polyclonal antibody C15410270 100 µg ELISA, Immunohistochemistry, WB Affinity purified
VPS72 polyclonal antibody C15200224 50 μg WB, IHC Protein A/G purified
ZEB1 polyclonal antibod C15410259 25 µg, 100 µg ChIP, ICC/IF, Immunoprecipitation, WB Affinity purified
ZHX2 polyclonal antibody C15410260 25 µl, 100 µl ChIP/ChIP-seq, ICC/IF, IHC, WB Affinity purified
ZNF189 polyclonal antibody C15410261 25 µg, 100 µg ICC/IF, WB Affinity purified
PIONEER Cat. No. Format Applications Purity
3-mC polyclonal antibody C15410209 50 μg Dot blot Protein G purified
ACTL6B polyclonal antibody C15310119 100 µl ELISA, WB Whole antiserum
AML-ETO polyclonal antibody C15410080 50 µg ELISA, WB Affinity purified
ASH2 polyclonal antibody C15310026 100 µl WB Whole antiserum
ASH2 polyclonal antibody C15310093 100 µl ELISA, WB, IF Whole antiserum
ASH2 polyclonal antibody C15410026 50 µg WB Protein G purified
BCL7C polyclonal antibody C15310124 100 µl ELISA, WB Whole antiserum
BMI1 polyclonal antibody C15310241 100 µl ELISA Whole antiserum
BRD2 polyclonal antibody C15310094 100 µl ELISA, WB Whole antiserum
CBX2 polyclonal antibody C15310239 100 µl ELISA Whole antiserum
CBX3 polyclonal antibody C15310234 100 µl ELISA Whole antiserum
CBX4 polyclonal antibody C15310251 100 µl ELISA Whole antiserum
CCDC101 polyclonal antibody C15310122 100 µl ELISA, WB Whole antiserum
CDH4 polyclonal antibody C15410031 50 µg WB Protein G purified
CDYL polyclonal antibody C15310243 100 µl ELISA Whole antiserum
CHAMP1 polyclonal antibody C15310250 100 µl ELISA Whole antiserum
Chd1 polyclonal antibody C15310020 100 µl WB Whole antiserum
www.diagenode.com | PAGE 28 DIAGENODE CHROMATIN IMMUNOPRECIPITATION
PIONEER Cat. No. Format Applications Purity
Chd1 polyclonal antibody C15410020 50 µg WB Protein G purified
CHD4 polyclonal antibody C15310031 100 µl WB Whole antiserum
CHD5 polyclonal antibody C15310121 100 µl ELISA, WB Whole antiserum
Cre polyclonal antibody C15310168 100 µl ELISA, WB Whole antiserum
ctr9/PAF1 polyclonal antibody C15310112 100 µl ELISA, WB Whole antiserum
DGCR8 polyclonal antibody C15310247 100 µl ELISA Whole antiserum
DNMT1 polyclonal antibody C15310077 100 µl ELISA, WB Whole antiserum
DNMT1 polyclonal antibody C15310111 100 µl ELISA, WB Whole antiserum
DNMT2 polyclonal antibody C15310095 100 µl ELISA, WB Whole antiserum
DNMT3A polyclonal antibody C15310107 100 µl ELISA, WB Whole antiserum
DNMT3B polyclonal antibody C15310044 100 µl ELISA, WB Whole antiserum
DNMT3B polyclonal antibody C15410218 50 μg ELISA, WB, IF Affinity purified
Dre polyclonal antibody C15310170 100 µl ELISA, WB Whole antiserum
DROSHA polyclonal antibody C15310246 100 µl ELISA Whole antiserum
EED polyclonal antibody C15410319 50 μg WB Protein G purified
EGFP polyclonal antibody C15410074 50 µg IF Affinity purified
EHMT1 polyclonal antibody C15310114 100 µl ELISA, WB Whole antiserum
EIF2C1/2 polyclonal antibody C15310141 100 µl ELISA, WB Whole antiserum
FKBP3 polyclonal antibody C15310109 100 µl ELISA, WB Whole antiserum
FKBP51 polyclonal antibody C15310110 100 µl ELISA, WB Whole antiserum
Flp polyclonal antibody C15310169 100 µl ELISA, WB Whole antiserum
G9a polyclonal antibody C15310096 100 µl ELISA, WB Whole antiserum
H1.0 polyclonal antibody C15310224 100 µl ELISA Whole antiserum
H1.1 polyclonal antibody C15310249 100 µl ELISA Whole antiserum
H1.3 polyclonal antibody C15310226 100 µl ELISA Whole antiserum
H1.4 polyclonal antibody C15310227 100 µl ELISA Whole antiserum
H1.5 polyclonal antibody C15310228 100 µl ELISA Whole antiserum
H2A.XS139p polyclonal antibody C15310223 100 µl ELISA Whole antiserum
H2Bs14p monoclonal antibody C15200186 50 μg ELISA, Dot blot Protein A purified
H3pan polyclonal antibody C15410059 50 µg ELISA, WB Affinity purified
H3pan polyclonal antibody C15310059 100 µl ELISA, IF Whole antiserum
H3T45p polyclonal antibody C15410321 50 µg ELISA, Dot blot Affinity purified
H3Y41p polyclonal antibody C15410320 50 μg ELISA, Dot blot Affinity purified
HDAC1 polyclonal antibody C15310042 100 µl WB Whole antiserum
Hira polyclonal antibody C15310097 100 µl ELISA, WB Whole antiserum
HMG2L1 polyclonal antibody C15310222 100 µl ELISA Whole antiserum
HP1α polyclonal antibody C15310070 100 µl WB Whole antiserum
HP1α polyclonal antibody C15410070 50 µg WB Protein G purified
Innovating Epigenetic Solutions PAGE 29
PIONEER Cat. No. Format Applications Purity
HP1α, ß & γ polyclonal antibody C15310071 100 µl WB Whole antiserum
Jarid1b polyclonal antibody C15310113 100 µl ELISA, WB Whole antiserum
Jarid1c polyclonal antibody C15310143 100 µl ELISA, WB Whole antiserum
JARID2 polyclonal antibody C15310235 100 µl ELISA Whole antiserum
JMJD2a polyclonal antibody C15410126 50 µg ELISA, WB Affinity purified
JMJD2b polyclonal antibody C15310104 100 µl ELISA, WB Whole antiserum
JMJD2c polyclonal antibody C15310105 100 µl ELISA, WB Whole antiserum
KDM1B polyclonal antibody C15310248 100 µl ELISA Whole antiserum
KDM4D polyclonal antibody C15310253 100 µl ELISA Whole antiserum
L3MBTL1 polyclonal antibody C15310023 100 µl WB Whole antiserum
Leo1 polyclonal antibody C15310017 100 µl WB Whole antiserum
Leo1 polyclonal antibody C15410017 50 µg WB Protein G purified
LRWD1 polyclonal antibody C15310232 100 µl ELISA Whole antiserum
LSD1 polyclonal antibody C15310028 100 µl WB Whole antiserum
LYDG10 polyclonal antibody C15310238 100 µl ELISA Whole antiserum m6A polyclonal antibody C15410208 50 μg Dot Blot Protein G purified
MBD1 polyclonal antibody C15410038 50 μg ELISA, WB Affinity purified
Mbd2 polyclonal antibody C15310098 100 µl ELISA, WB Whole antiserum
MBD3 polyclonal antibody C15310079 100 µl ELISA, WB Whole antiserum
MBD4 polyclonal antibody C15310087 100 µl ELISA, WB Whole antiserum
MeCP2 polyclonal antibody C15310088 100 µl ELISA, WB Whole antiserum
MeCP2 polyclonal antibody C15310106 100 µl ELISA, WB Whole antiserum
MeCP2 polyclonal antibody C15410217 50 μg WB Protein G purified
Med26 polyclonal antibody C15310073 100 µl WB Whole antiserum
Med26 polyclonal antibody C15410073 50 µg WB Protein G purified
Mll2 polyclonal antibody C15310099 100 µl ELISA, WB Whole antiserum
Mll4 polyclonal antibody C15310100 100 µl ELISA, WB Whole antiserum
MTA2 polyclonal antibody C15410032 50 µg WB Protein G purified
PADI4 polyclonal antibody C15310075 100 µl ELISA, WB Whole antiserum
Paf1 polyclonal antibody C15310015 100 µl WB Whole antiserum
PAPD1 polyclonal antibody C15310132 100 µl ELISA, WB Whole antiserum
PAPD4 polyclonal antibody C15310130 100 µl ELISA, WB Whole antiserum
PAPD5 polyclonal antibody C15310134 100 µl WB Whole antiserum
PAPOLA polyclonal antibody C15310137 100 µl ELISA, WB Whole antiserum
PHC2 polyclonal antibody C15310240 100 µl ELISA Whole antiserum
PHF8 polyclonal antibody C15410064 50 µg ELISA, WB Affinity purified
PIWIL1 polyclonal antibody C15310244 100 µl ELISA Whole antiserum
PIWIL2 polyclonal antibody C15310252 100 µl ELISA Whole antiserum
www.diagenode.com | PAGE 30 DIAGENODE CHROMATIN IMMUNOPRECIPITATION
PIONEER Cat. No. Format Applications Purity
PML polyclonal antibody C15410108 50 µg ELISA, WB Affinity purified
RbAp 46/48 monoclonal antibody C15100033 100 µl WB Ascites fluid
RbAp 46/48 monoclonal antibody C15200033 50 µg WB Protein G purified
SAP30 polyclonal antibody C15310036 100 µl WB Whole antiserum
SAP30 polyclonal antibody C15410036 50 µg WB Protein G purified
Set9 polyclonal antibody C15310047 100 µl WB Whole antiserum
Set9 polyclonal antibody C15410047 50 µg WB Protein G purified
Setd1a polyclonal antibody C15310117 100 µl ELISA, WB Whole antiserum
Setd1b polyclonal antibody C15310118 100 µl ELISA, WB Whole antiserum
SETD8 polyclonal antibody C15310043 100 µl WB Whole antiserum
SETD8 polyclonal antibody C15410043 50 µg WB Protein G purified
SirT1 monoclonal antibody C15100063 100 µl WB Ascites fluid
SirT1 monoclonal antibody C15200063 50 µg WB Protein G purified
Smc3K112/113ac polyclonal C15310229 100 µl ELISA Whole antiserum antibody
SOX4 polyclonal antibody C15310129 100 µl ELISA, WB Whole antiserum
SPAST monoclonal antibody C15200001 50 µg WB Affinity purified
SPI1 polyclonal antibody C15310120 100 µl ELISA, WB Whole antiserum
Spt16 polyclonal antibody C15310049 100 µl WB Whole antiserum
Spt16 polyclonal antibody C15410049 50 µg WB Protein G purified
SSRP1 polyclonal antibody C15310050 100 µl WB Whole antiserum
SSRP1 polyclonal antibody C15410050 50 µg WB Protein G purified
TAF1 polyclonal antibody C15310072 100 µl WB Whole antiserum
TASP1 polyclonal antibody C15310115 100 µl ELISA, WB Whole antiserum
WDR5 polyclonal antibody C15310027 100 µl WB Whole antiserum
WDR5 polyclonal antibody C15310101 100 µl ELISA, WB Whole antiserum
WDR5 polyclonal antibody C15410027 50 µg WB Protein G purified
YY1 polyclonal antibody C15310242 100 µl ELISA Whole antiserum
ZBED6 polyclonal antibody C15310237 100 µl ELISA Whole antiserum
ZBTB38 polyclonal antibody C15310123 100 µl ELISA, WB Whole antiserum
ZBTB4 polyclonal antibody C15310142 100 µl ELISA, WB Whole antiserum
ZMYND8 polyclonal antibody C15310136 100 µl ELISA, WB Whole antiserum
OTHER ANTIBODIES Cat. No. Format Applications Purity
Blue ladder - HRP monoclonal C15200202-10 10 µl WB Protein A purified antibody C15200202-100 100 µl WB Protein A purified
Blue ladder monoclonal antibody C15200213 50 µl WB Protein A purified
Innovating Epigenetic Solutions PAGE 31
Our latest groundbreaking Industry-leading ChIP and ChIP-seq innovation – ChIP-seq with kits 10,000 cells With an exclusive focus on epigenetics, Diagenode For ChIP-seq with limited sample amounts, the solution of provides the highest quality epigenetics products on choice is the combination of the True MicroChIP kit with the the market. Our complete suite of ChIP and ChIP-seq MicroPlex Library Preparation™ kit and our Premium grade solutions includes extensively validated ChIP-seq antibodies, enabling ChIP-seq with just a few picograms grade Premium antibodies, cell lysis and chromatin ® of input. The MicroPlex kit is an exceptionally fast, user- shearing devices (Bioruptor ), kits for high and friendly, and automatable solution with multiplexing low cell numbers, automated epigenetics sample capability optimized for ChIP-seq libraries from picogram preparation, and kits specialized for ChIP-seq. Our inputs of chromatin. The library preparation requires only optimized, high performance ChIP kits are the result three simple steps, with no tube transfers or intermediate of over 10 years of research and development. We purifications required. Additionally, the True MicroChIP kit provide a flexible platform that addresses specific has been validated on the Illumina® sequencing platforms, research needs with a vast array of ChIP kits Diagenode’s IP-Star® Compact Automated Workstation, optimized for a range of high cell amounts or very and with our Premium ChIP-seq grade antibodies, the most low cell amounts down to 200 cells per IP. We also extensively validated antibodies on the market selected have developed groundbreaking kits for ChIP-seq specifically for ChIP-seq applications. starting with inputs of only 10,000 cells. Our ChIP- seq kits have been specifically optimized on the most commonly used sequencing systems (Illumina® GAIIx and Ion Torrent™ PGM™).
True MicroChIP Kit and MicroPlex Library True MicroChIP kit Bioruptor® Pico Preparation™ Kit Revolutionary: Highly efficient ChIP recovery (1) and enrichment from only 10,000 cells Reliable and accurate sequencing library preparation from just picogram inputs Simple three-step library prep protocol with NO intermediate purifications!
(2) Automatable with the IP-Star for highest consistency
(3) a. b.(4) c. (5) d. (6)
a. b. c. d. a. b. c. d.
Figure 1: True MicroChIP procedure: 1. Cell fixation and DNA-protein cross-linking, 2. Cell lysis and chromatin shearing using the Bioruptor®, 3. Binding of the antibodies to the chromatin, addition of beads, 4. Magnetic immunoprecipitation, 5. Washes of immune complexes, 6. DNA purification and recovery of ChIP’d DNA; ChIP-sequencing procedure: 1. End-repair, 2. Adapter ligation, 3. Library amplification, 4. Library purification and sequencing on Illumina® platform.
www.diagenode.com | PAGE 32 DIAGENODE CHROMATIN IMMUNOPRECIPITATION
Which ChIP kit is right?
CHIP OF CHIP OF TRANSCRIPTION LOW INPUT CHIP HISTONES FACTORS PLANT CHIP
iDeal ChIP-seq Kit iDeal ChIP-seq Kit for True MicroChIP Kit for Histones Transcription Factors Plant ChIP-seq Kit (10k - 100k cells) (100k - 1 million cells) (4 million cells)
qPCR/Microarray Library Preparation
Which Library Preparation Kit is right?
LOW INPUT HIGH INPUT
MicroPlex Library iDeal Library Preparation Kit Preparation Kit (50 pg - 5 ng input) (> 5 ng input)
Sequencing
Innovating Epigenetic Solutions PAGE 33
Manual ChIP Kits iDeal ChIP-seq Kit for Histones
Validated: Only kit on the market validated for GAIIx (Illumina®) and PGM™ (Ion Torrent™) Proven: Our expertise with ChIP-seq tools enables reproducible and efficient results every time Complete: Kit includes control antibodies, control primer pairs and IPure DNA purification module
Figure 1A shows a several hundred bp stretch A. along the 12th chromosome, where the high similarity of read distribution can be observed, despite the radically different characteristics of the sequencers. ChIP was performed on chromatin from HelaS3 cells using 1 µg of H3K4me3 antibody.
B. Figure 1B is a close capture focusing on the well-known GAPDH gene. The detailed view reveals that even the peak structure is similar.
Figure 2: ChIP-seq has been performed using chromatin from HeLa cells, the iDeal ChIP-seq kit and the Diagenode ChIP-seq-grade H3K4me3 antibody. The ChIP-seq dataset from this experiment has been compared with a reference dataset from the Broad Institute. We observed a perfect match between the top 40% of Diagenode peaks and the reference dataset. Based on the NIH Encode project criterion, ChIP-seq results are considered reproducible between an original and reproduced dataset if the top 40% of peaks have at least an 80% overlap ratio with the compared dataset.
Product Cat. No. Format
iDeal ChIP-seq kit for Histones x10 C01010050 10 rxns
iDeal ChIP-seq kit for Histones x24 C01010051 24 rxns
iDeal ChIP-seq kit for Histones x100 C01010059 100 rxns iDeal ChIP-seq & Library iDeal ChIP-seq kit for Histones Preparation Package (incl. Index C01010053 24 rxns Primer Set 1)
* Pinpoint exact transcription factor binding sites
www.diagenode.com | PAGE 34 DIAGENODE CHROMATIN IMMUNOPRECIPITATION
iDeal ChIP-seq Kit for Transcription Factors
Confidence in results: Validated for ChIP-seq with multiple transcription factors Proven: Validated by the epigenetics community, including the BLUEPRINT consortium Most complete kit available for highest quality data -- includes controls, primers, and magnetic beads Validated with Diagenode’s MicroPlex Library Preparation Kit and IP-Star® Automation
A. Figure 1A: Chromatin Immunoprecipitation has been performed using chromatin from HeLa cells, the iDeal ChIP-seq kit for Transcription Factors and the Diagenode ChIP-seq-grade CTCF antibody. The IP’d ® CTCF DNA was subsequently analysed on an Illumina HiSeq. Diagenode Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. This figure shows the peak distribution in CTCF a region surrounding the GAPDH positive control gene. Encode
B. Total Diagenode peaks Overlapping peaks Figure 1B: The ChIP-seq dataset from this experiment with Encode dataset has been compared with a reference dataset from the Broad Institute. We observed a perfect match between the top 40% of Diagenode peaks and the reference 95% dataset. Based on the NIH Encode project criterion, 40% ChIP-seq results are considered reproducible between an original and reproduced dataset if the top 40% of peaks have at least an 80% overlap ratio with the compared dataset.
A. Figure 2: Chromatin Immunoprecipitation HDAC1 has been performed using chromatin from HeLa cells, the iDeal ChIP-seq kit for Transcription Factors and the Diagenode ChIP-seq-grade HDAC1 (A), LSD1 (B) and p53 antibody (C). The IP’d B. DNA was subsequently analysed on an LSD1 Illumina® Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. This figure shows the peak distribution C. in regions of chromosome 3 (A), p53 chromosome 12 (B) and chromosome 6 (C) respectively.
Product Cat. No. Format
iDeal ChIP-seq Kit for Transcription Factors x10 C01010054 10 rxns
iDeal ChIP-seq Kit for Transcription Factors x24 C01010055 24 rxns
iDeal ChIP-seq Kit for Transcription Factors x100 C01010170 100 rxns iDeal ChIP-seq for Transcription Factors & 10 ChIP & 12 library C01010056 iDeal ChIP-seq kit for Transcription Factors MicroPlex Library Preparation™ Package prep rxns
Innovating Epigenetic Solutions PAGE 35
True MicroChIP Kit
Only kit that allows for ChIP-seq on 10,000 cells
Revolutionary: Only 10.000 cells needed for complete ChIP-seq procedure Reliable and accurate sequencing library preparation from just picogram inputs Validated on the IP-Star® Compact Automated Platform
Figure 1: ChIP has been peformed with H3K4me3 antibody, amplification of 17 pg of DNA ChIP’d from 10.000 cells and amplification of 35 pg of DNA ChIP’d from 100.000 cells (control experiment). The IP’d DNA was amplified and transformed into a sequencing- ready preparation for the Illumina plateform with the MicroPlex Library Preparation kit. The library was then analysed on an Illumina® Genome Analyzer. Cluster generation and sequencing were performed according to the manufacturer’s instructions.
Figure 2: We observed a perfect match between the top 40% of True MicroChIP peaks and the reference dataset. Based on the NIH Encode project criterion, ChIP-seq results are considered reproducible between an original and reproduced dataset if the top 40% of peaks have at least an 80% overlap ratio with the compared dataset.
Product Cat. No. Format
True MicroChIP kit C01010130 16 rxns True MicroChIP & MicroPlex Library Preparation™ 16 ChIP rxns and 12 C01010131 Package library prep rxns
True MicroChIP kit
www.diagenode.com | PAGE 36 DIAGENODE CHROMATIN IMMUNOPRECIPITATION
Plant ChIP-seq Kit
Optimized for extracting plant chromatin Complete kit - includes plant-specific controls and primers Validated with low DNA input library preparation kit Compatible with high throughput IP-Star® Automated System
Figure 1: ChIP-seq was performed on Arabidopsis thaliana (Col-0) seedlings using our Premium H3K4me3 ChIP-seq grade antibody (Cat. No. 1 ng C15410003). Libraries were prepared with our MicroPlex Library Preparation™ kit (Cat. No.
500 pg C05010010) from 1 ng (green), 500 pg (orange) and 100 pg (red) IP’d DNA and sequenced on an Illumina® HiSeq 2500. The enrichment in blue 100 pg represents public dataset (NCBI GEO Dataset GSM1193621) that we used as an external reference. Enrichments along a wide region of Reference dataset chromosome 5 are uniform regardless of the starting material amount for the preparation of the library.
5 FLC ATG (pos.) Figure 2: ChIP was performed on Arabidopsis thaliana (Col-0) seedlings 14 days after germination (dag) using our premium anti-H3K4me3 ChIP- 4 FLC Intron (neg.) seq grade antibody (#C15410003) and rabbit IgG (#C15410206). Data shown is derived from biological replicates. Sheared chromatin from 0.2 g (fw) of
3 seedlings, 1 μg of H3K4me3 antibody and 1 μg of the negative IgG control were used per IP. Quantitative PCR was performed with the positive control FLC-ATG and the negative control FLC-Intron1 primer sets from the kit. The 2 recovery is expressed as % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). 1
0 H3K4me3 IgG
1.5 FLC intron1 (pos.) Figure 3: ChIP was performed on Arabidopsis thaliana (Col-0) seedlings 14 days after germination (dag) using our premium anti-H3K27me3 ChIP- Actin ATG (neg.) seq grade antibody (#C15410195) and rabbit IgG (#C15410206). Data shown is derived from biological replicates. Sheared chromatin from 0.2 g (fw) of 1.0 seedlings, 2 μg of H3K27me3 antibody and 2 μg of the negative IgG control were used per IP. Quantitative PCR was performed with primer pairs for FLC- Intron1 (positive control) and Actin ATG (negative control). The recovery is
0.5 expressed as % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).
0.00 Product Cat. No. Format H3K27me3 IgG Plant ChIP-seq Kit x24 C01010150 24 rxns
I was using the Diagenode Plant ChIP-seq kit to investigate abundance of particular histone mark in A.thaliana’s seedlings. The Plant ChIP-seq kit yielded high amounts of immunoprecipitated DNA, very good relative enrichment to input and low background level. I frankly recommend it for ChIP on plant tissues.
Msc Pawel Mikulski, Lab of Dr. Daniel Schubert, Heinrich Heine University Düsseldorf
Innovating Epigenetic Solutions PAGE 37
Which Auto ChIP kit is right?
CHIP OF CHIP OF TRANSCRIPTION LOW INPUT CHIP HISTONES FACTORS PLANT CHIP
Auto iDeal ChIP-seq Kit Auto iDeal ChIP-seq Kit Auto True MicroChIP Kit for Histones for Transcription Factors Auto Plant ChIP-seq Kit (10k - 100k cells) (100k - 1 million cells) (4 million cells)
qPCR/Microarray Library Preparation
Which Library Preparation Kit is right?
LOW INPUT HIGH INPUT
MicroPlex Library iDeal Library Preparation Kit Preparation Kit (50 pg - 5 ng input) (> 5 ng input)
Sequencing
IP-Star® Compact Automated System
www.diagenode.com | PAGE 38 DIAGENODE CHROMATIN IMMUNOPRECIPITATION
Kits for ChIP on the IP-Star® Compact Automated System
Auto True MicroChIP Kit Auto iDeal ChIP-seq kit for Transcription Factors
Compatible with 5,000-100,000 cells per IP 4 millions cells per IP Validated for histone antibodies Validated for ChIP experiments with antibodies directed to transcription factors, epitope-tagged Ideal for whole genome sequencing from low amount transcription. of chromatin when combined with the MicroPlex Library Prep Kit
Chromatin Immunoprecipitation using chromatin from HeLa cells, the iDeal ChIP-seq kit for Transcription Factors and the Diagenode ChIP-seq-grade CTCF antibody. The IP’d DNA Library generation with the MicroPlex kit. ChIP assays were was subsequently analysed on an Illumina® HiSeq. Library performed on 10,000 and 100,000 HeLa cells with Diagenode preparation, cluster generation and sequencing were performed H3K4me3 antibody (0.25 µg/reaction). Libraries were made according to the manufacturer’s instructions. This figure shows with the MicroPlex Library Preparation Kit. The generated the peak distribution in a region surrounding the GAPDH positive libraries were then analyzed on an Illumina® HiSeq2000. Cluster control gene. generation and sequencing were performed according to the manufacturer’s instructions.
Product Cat. No. Format
Auto True MicroChIP kit C01010140 16 rxns 16 ChIP and Auto True MicroChIP & MicroPlex C01010141 12 library prep Library Preparation Package rxns Auto iDeal ChIP-seq kit for Histones
1 million cells per IP Validated for ChIP-seq and ChIP-qPCR experiments Chromatin Immunoprecipitation has been performed using chromatin from HeLa cells, the iDeal ChIP-seq kit for with antibodies directed to histone modifications Transcription Factors and the Diagenode ChIP-seq-grade Ideal for whole genome sequencing HDAC1 (A), LSD1 (B) and p53 antibody (C). The IP’d DNA was ® Image A shows a several subsequently analysed on an Illumina Genome Analyzer. hundred bp along chr12 Library preparation, cluster generation and sequencing were with high similarity of performed according to the manufacturer’s instructions. This read distribution despite figure shows the peak distribution in regions of chromosome 3 the radically different (A), chromosome 12 (B) and chromosome 6 (C) respectively. sequencers.
Image B is a close Product Cat. No. Format capture focusing on Auto iDeal ChIP-seq Kit for the GAPDH that shows C01010058 24 rxns Transcription Factors x24 that even the peak Auto iDeal ChIP-seq Kit for structure is similar. C01010172 100 rxns Transcription Factors x100
Product Cat. No. Format
Auto iDeal ChIP-seq kit for Histones x24 C01010057 24 rxns
Auto iDeal ChIP-seq kit for Histones x100 C01010171 100 rxns
Innovating Epigenetic Solutions PAGE 39
Auto Plant ChIP-seq Kit
Optimized for extracting plant chromatin Complete kit - includes plant-specific controls and primers Validated with low DNA input library preparation kit
A. ChIP-seq was performed on Arabidopsis Thaliana (Col-0) seedlings using our Premium H3K4me3 ChIP-seq grade antibody (Cat. No. C15410003). Libraries were prepared with our MicroPlex Library Preparation™ Kit (Cat. No. C05010010) from 1 ng (green), 500 pg (orange) and 100 pg (red) IP’d DNA and sequenced on an Illumina® HiSeq 2500. The enrichment in blue represents public dataset (NCBI BEO Dataset GSM1193621) that we used as an external reference. Enrichments along a wide region of chromosome 5 are uniform regardless of the starting material amount for the preparation of the library.
Product Cat. No. Format
Auto Plant ChIP-seq kit x24 C01010151 24 rxns
DiaMag Product Range The DiaMag Racks and Rotator simplify your workflow without the DiaMag magnetic racks – fast, efficient isolation of magnetic need to work with individual tubes beads in ChIP DiaMag Magnetic Beads - extensively validated for ChIP 1. Place disc holder DiaMag Rotator – seamless integration into your workflow with tubes into rack. DiaMag beads Description Cat. No. adhere to the tube DiaMag magnetic racks and rotator wall.
DiaMag02 - magnetic rack B04000001
DiaMag1.5 magnetic rack & disc stand B04000002 2. Remove the DiaMag1.5 - magnetic rack B04000003 buffer with pipet without disrupting DiaMag1.5 - disc stand B04000004 immobilized magnetic bead DiaMag Rotator B05000001 complex. DiaMag protein- or antibody-coated beads
DiaMag protein G-coated magnetic beads 3. Take off disc with Compatible with mouse IgG1, IgG2a, IgG2b and IgG3, C03010021-220/660/150/005 tubes and place rat IgG1, IgG2a, IgG2b and IgG3 rabbit and goat (220 µl, 660 µl, 1.5 ml , 5 ml) polyclonal Abs and human IgG1, IgG2, IgG3 and IgG4 onto disc stand. Resuspend sample DiaMag protein A-coated magnetic beads C03010020-220/660/150/005 with wash buffer. Compatible with rabbit polyclonal Abs, mouse IgG2a, (220 µl, 660 µl, 1.5 ml , 5 ml) IgG2b and IgA, guinea pig IgG, dog IgG, pig IgG
DiaMag anti-mouse IgG-coated magnetic beads. C03010022-220/660/150 4. Put disc with tubes Compatible with mouse IgG1, IgG2a, IgG2b (220 µl, 660 µl, 1.5 ml) on the DiaMag Rotator. For multiple washes, repeat steps 1-4.
www.diagenode.com | PAGE 40 DIAGENODE CHROMATIN IMMUNOPRECIPITATION
ChIP kits
Manual kits Automated kits iDeal ChIP- Auto iDeal Auto iDeal True MicroChIP iDeal ChIP-seq seq Kit for Auto True ChIP-seq kit for ChIP-seq Kit for kit Kit for Histones Transcription MicroChIP kit Transcription Histones Factors Factors Low cell numbers • Histone • Transcription Low cell numbers • Histone • Transcription from 10,000 to targets Factor targets from 10,000 to targets Factor targets Optimized for 100,000 cells/IP • 100,000 to 1 • 1 million to 4 100,000 cells/IP • 100,000 to 1 • 1 million to 4 million cells/IP million cells/IP million cells/IP million cells/IP MicroPlex Library • MicroPlex • MicroPlex MicroPlex Library • MicroPlex • MicroPlex Preparation Kit Library Library Preparation Kit Library Library (50 pg - 5 ng Preparation Kit Preparation Kit (50 pg - 5 ng Preparation Kit Preparation Kit Recommended input) (50 pg - 5 ng (50 pg - 5 ng input) (50 pg - 5 ng (50 pg - 5 ng library input) input) input) input) prepartion kit for • iDeal Library • High • iDeal Library • High ChIP-seq Preparation Kit resolution Preparation Kit resolution (> 5 ng input) Library (> 5 ng input) Library Preparation kit Preparation kit Suitable for qPCR/ microarrays/ Yes Yes Yes Yes Yes Yes sequencing Time from cell 2 days 2 days 2 days 1 day 1 day 1 day collection to PCR 10, 24 or 100 10, 24 or 100 24 or 100 24 or 100 Format 16 reactions 16 reactions reactions reactions reactions reactions Lysis, chromatin Lysis, chromatin Lysis, chromatin Lysis, chromatin Lysis, chromatin Lysis, chromatin Included shearing, shearing, shearing, shearing, shearing, shearing, reagents for Immuno- Immuno- Immuno- Immuno- Immuno- Immuno- precipitation precipitation precipitation precipitation precipitation precipitation • Control IgG • Control IgG • Control IgG • Control IgG • Control IgG • Control IgG Control • ChIP-seq • ChIP-seq • ChIP-seq • ChIP-seq • ChIP-seq • ChIP-seq antibodies grade grade grade CTCF grade grade grade CTCF H3K4me3 H3K4me3 H3K4me3 H3K4me3 • ChIP-seq • ChIP-seq • ChIP-seq • ChIP-seq • ChIP-seq • ChIP-seq grade GAPDH grade GAPDH grade H19 grade GAPDH grade GAPDH grade H19 TSS TSS imprinting TSS TSS imprinting Control PCR • ChIP-seq • ChIP-seq control region • ChIP-seq • ChIP-seq control region primer pairs grade grade • ChIP-seq grade grade • ChIP-seq myoglobin myoglobin grade myoglobin myoglobin grade exon 2 exon 2 Myoglobin exon 2 exon 2 Myoglobin exon 2 exon 2 PCIA (optional: IPure (magnetic IPure (magnetic Phenol/ IPure (magnetic IPure (magnetic DNA purification MicroChIP DiaPure bead-based) bead-based) chloroform/ bead-based) bead-based) columns) isoamyl alcohol)
Library Preparation Kits
MicroPlex Library Preparation kit v2 iDeal Library Preparation kit
• Validated with the IP-Star® Automated Platform • Validated with the IP-Star® Automated Platform • Specifically optimized for ChIP-seq • Multiplexing capacity of up to 24 samples Features • 100x more sensitive than other methods with only • Optimized with iDeal ChIP-seq Kit for ChIP picogram inputs • Simple three step protocol with NO intermediate purifications Recommended 50 pg to 5 ng 5 ng - 1 µg input amounts • Low input ChIP-seq • High input ChIP-seq Application • Low input DNA sequencing • Genomic DNA sequencing • Transcription Factor ChIP-seq Multiplexing 12 or 48 indices 12 indices (12 additional indices - optional)
Innovating Epigenetic Solutions PAGE 41
Overview Automated Solutions Diagenode's Automated Platforms replace the numerous manual, error-prone steps of complex epigenetic applications Chromatin Immunoprecipitation combined with with a reliable, highly consistent and automated process massively parallel sequencing (ChIP-seq) has become that requires minimal operator intervention. Diagenode's the gold standard for whole-genome mapping Automated Systems minimize sample carryover, data of protein-DNA interactions. The reproducibility variability, and costly errors. The platforms offer full workflow and biological relevance of DNA associated support for epigenetics research, utilizing our complete kits protein landscapes depend on the specificity and and laboratory-validated protocols to rapidly deliver high- performance of the antibodies and optimized quality and consistent data. protocols are needed to ensure high recovery and increased signal-to-noise ratio. Automation of such Diagenode’s Automated System uses the principle of bead- approaches is required to achieve lower cost/sample, based magnetic separation. Magnetic beads bound with and higher reproducibility and to increase lab chromatin or DNA are brought to the inner wall of the tip productivity. Diagenode’s SX-8G IP-Star® Automated when a strong magnetic force is applied. This differs from Systems enhance, in combination with Diagenode’s other systems that collect the bound DNA on the bottom of a whole range of kits, the reproducibility needed for the reaction well, resulting in cleaner assays and less carryover. ChIP-seq assays allowing the researcher to optimize multiple parameters which are critical for reliable and robust genome wide profiling
Additional applications Chromatin Immunopreocipitation (ChIP) Methylated DNA Immunoprecipitation (MeDIP) Hydroxymethylated DNA Immunoprecipitation (hMeDIP) Methyl-binding domain protein capture assays (MethylCap) Sequential ChIP (Re-ChIP) Bisulfite Conversion RNA-Immnunoprecipitation (RNA-IP) DNA purification (IPure) Library preparation for NGS platforms
8 Channel pipettor
Magnetic bar
Tips for reagents and incubation
Peltier block www.diagenode.com | PAGE 42 DIAGENODE CHROMATIN IMMUNOPRECIPITATION
Walk-away automated system
Magnetic-bead technology for excellent Processing of to 16 samples reproducibility Protocols optimal for 100 µl or 200 µl volumes Open platform allows changes in protocol parameters Automated reagent dispensing Simple touch screen interface Controlled temperatures via Peltier blocks Minimal footprint
Overview of the Automated System
8-channel pipetting unit
96 tips module 96 tips module Peltier block 1 Peltier block 2
Reagent container module Reagent container 96 well plate module 96 well plate module module
Dustbin
The Magtration® Technology Diagenode’s Automated System uses the principle of bead-based magnetic separation. Magnetic beads bound with chromatin or DNA are brought to the inner wall of the tip when a strong magnetic force is applied. This differs from other systems Magnetic bar that collect the bound DNA on the bottom of a reaction well, resulting in cleaner assays and less carryover.
Innovating Epigenetic Solutions PAGE 43
ChIP Applications
High Sensitivity Transcription Factor ChIP
A 02 PTK2 Peak3 B 02 PTK2 Peak3 0.7 250
0.6 200 0.5
0.4 150 0.3 100 % of input 0.2 Fold enrichment 0.1 50 0 01 Goat 02 ETO 03 Goat 04 ETO 05 Goat 06 ETO 0 Mock Mock AGF6 AGF6 AGF1 AGF1 02 ETO Mock 04 ETO AGF6 06 ETO AGF1 C 03 PTK2 Peak4 D 03 PTK2 Peak4
0.07 350
0.06 300
0.05 250
0.04 200 0.03 150 % of input 0.02 100 0.01 Fold enrichment 50 0 01 Goat 02 ETO 03 Goat 04 ETO 05 Goat 06 ETO 0 Mock Mock AGF6 AGF6 AGF1 AGF1 02 ETO Mock 04 ETO AGF6 06 ETO AGF1
ChIP data from AML1-ETO antibody in two different genomic regions on the PTK2 promoter region. ChIP analysis of ET0 and control IgG antibody in SKNO-1 cells treated with a siRNA that specifically targets the fusion site in AML1- ETO (siAGF1), a mismatch control (siAGF6) and cells without siRNA treatment (Mock). In the left diagrams (A and C), results are shown in % of input. On the right panel, results are shown as fold enrichment versus the control antibody for the three different siRNA conditions.
Automated ChIP experiments in the IP-Star® Compact Automated System can be used to detect changes in transcription factor binding after treatment with small interference RNAs. Compared to conventionnal ChIP, the use of the SX-8G IP-Star® Compact in combination with the Auto ChIP kit saves working time and improves the reproducibility of ChIP experiments. S.Prall and O. Heidenreich, Northern Institute of Cancer Research, Newcastle University. C. Bonifer, LIMM, University of Leeds
Full walk-away automation to generate ChIP-seq profiles genome-wide
Scale 50 Mb hg18 Figure shows the profiles obtained in ChIP- chrX: 50,000,000 100,000,000 150,000,000 422 - H3K4me3 seq on the IP-Star automated system with
1 _ 239 - antibodies against (1) H3K4me3, H3K9ac H3K9ac and H3K9/14ac, histone modifications 1 _ 286 - that are present at active promoters, (2) H3K9/14ac 1 _ H3K4me2 which is surrounding promoters 73 - H3K4me2 of active genes, (3) H3K79me3 present at 1 _ 146 - promoters of active genes and extending H3K79me3
1 _ into the gene body, and (4) H3K36me3, 76 - H3K36me3 present at the gene bodies of active genes.
1 _ RefSeq Genes The screenshot shows the peak distribution along the complete X-chromosom.
www.diagenode.com | PAGE 44 DIAGENODE CHROMATIN IMMUNOPRECIPITATION
Library Preparation protocols
Standard Library Preparation protocols End repair Quick and easy set up, 15 minutes hands-on time AMPure purification Quick and easy set up, 15 minutes hands-on time Automated in the IP-Star Compatible with multiplexing protocols Adenylation Process 16 samples per run, 32 samples per day
Clean-up of adaptors and size selection using AMPure XP beads Adapter ligation PCR amplification is optional Recommended with Bioruptor® for DNA fragmentation Stop ligation ®
Available protocols AMPure purification Illumina® TruSeq™ ChIP Illumina® TruSeq™ DNA AMPure Size selection Illumina® TruSeq™ DNA PCR-Free PCR amplification (optional) NEBNext® ChIP-Seq Library Prep for Illumina® Illumina® TruSeq™ RNA Sequencing Illumina® TruSeq™ Stranded RNA
SX-8G IP-STAR® Reference Papers THP1 cells Apr 1;9(4):546-56. Sharma G, Upadhyay S, Srilalitha M, Nandicoori VK, Khosla S, The interaction of mycobacterial protein Rv2966c with host chromatin is Human and chimpanzees peripheral blood mediated through non-CpG methylation and histone H3/H4 binding. Wilson GA, Butcher LM, Foster HR, Feber A, Roos C, Walter L, Woszczek Nucleic Acids Res. 2015 Mar 30 G, Beck S, Bell CG, Human-specific epigenetic variation in the immunological Leukotriene B4 Receptor (LTB4R/BLT1) implicated in Plants - Arabidopsis common inflammatory diseases. Genome Med. 2014 Mar 5;6(3):19. Ariel F, Jegu T, Latrasse D, Romero-Barrios N, Christ A, Benhamed M, Crespi M., Noncoding Transcription by Alternative RNA Polymerases Embryonic stem cells Dynamically Regulates an Auxin-Driven Chromatin Loop. Mol Cell. Veillard AC, Marks H, Bernardo AS, Jouneau L, Laloe D, Boulanger L, 2014 Jul 9. Kaan A, Brochard V, Tosolini M, Pedersen R, Stunnenberg H, Jouneau A. Stable methylation at promoters distinguishes Epiblast Stem Cells Peripheral/induced T regulatory cells from Embryonic Stem Cells and the in vivo epiblast. Stem Cells Dev. Sarmento OF, Svingen PA, Xiong Y, Xavier RJ, McGovern D, Smyrk TC, 2014 Apr 16. Papadakis KA, Urrutia RA, Faubion WA.A novel role for KLF14 in T regulatory cell differentiation. Cell Mol Gastroenterol Hepatol. 2015 Human – prostate cancer Mar 1. Ngollo M, Dagdemir A, Judes G, Kemeny JL, Penault-Llorca F, Boiteux JP, Lebert A, Bignon YJ, Guy L, Bernard-Gallon D, Epigenetics of RNA immunoprecipitation prostate cancer: distribution of histone H3K27me3 biomarkers in Carlotto N, Wirth S, Furman N, Ferreyra Solari N, Ariel F, Crespi M, peri-tumoral tissue., OMICS,2014-03-01,18,207-9. Kobayashi K, The chloroplastic DEVH-box RNA helicase INCREASED SIZE EXCLUSION LIMIT 2 involved in plasmodesmata regulation is Human – blood samples required for group II intron splicing. Plant Cell Environ. 2015 Jul 4. Bell JT, Loomis AK, Butcher LM, Gao F, Zhang B, Hyde CL, Sun J, Wu H, Ward K, Harris J, Scollen S, Davies MN, Schalkwyk LC, Mill J; MuTHER Mice - Thymocytes Consortium, Williams FM, Li N, Deloukas P, Beck S, McMahon SB, Haljasorg U, Bichele R, Saare M, Guha M, Maslovskaja J, Kõnd K, Wang J, John SL, Spector TD. Differential methylation of the TRPA1 Remm A, Pihlap M, Tomson L, Kisand K, Laan M, Peterson P., A promoter in pain sensitivity. Nat Commun. 2014;5:2978. doi: 10.1038/ highly conserved NF-κB-responsive enhancer is critical for thymic ncomms3978. expression of Aire in mice. Eur J Immunol. 2015 Dec. Bacteria Cell lines Pacis A, Tailleux L, Morin AM, Lambourne J, MacIsaac JL, Yotova V, Laget S, Miotto B, Chin HG, Estève PO, Roberts RJ, Pradhan S, Defossez Dumaine A, Danckaert A, Luca F, Grenier JC, Hansen KD, Gicquel B, Yu M, PA. MBD4 cooperates with DNMT1 to mediate methyl-DNA repression Pai A, He C, Tung J, Pastinen T, Kobor MS, Pique-Regi R, Gilad Y, Barreiro and protects mammalian cells from oxidative stress. Epigenetics. 2014 LB. Bacterial infection remodels the DNA methylation landscape of human dendritic cells. Genome Res. 2015 Dec.
Innovating Epigenetic Solutions starting material (input)usedranges from 5ngto 1µgofDNA. Sample I has been purified in duplicates using the IPure kit v2. The No. (Cat. kit ChIP LowCell# quantified withaNanodrop. amounts the different C15410056; 2 No. (Cat. antibody cells, using H3K9me3 the and C01010070) perfomed U2OS were of assays ChIP technology IPure using samples ChIP of purification after recovery DNA extracting very low amountsofDNAafter ChIPandMeDIP. The only DNA purification kit that is specifically optimized for v2 IPure Kit Auto IPure kitv2x100 IPure kitv2x100 IPure kitv2x24 Product g/IP). The purified DNA was eluted in50 eluted was DNA purified The μg/IP). DNA recovery (ng)
10 12 14 16 18 6 8 4 2 0 Toxic reagents notused(e.g.phenol/chloroform) (e.g. Next generation sequencing) Pure DNAobtained for anydownstream application Straightforward protocol usingmagneticbeads Recovery ofsmallamountsDNA purification Much higheryieldthanwithcolumn-based 1,000 c 0.2 ng ells 10,000 c 1.96 ng ells 100,000 c C03010010 C03010015 C03010014 Cat. No. 12.95 ng ells o wtr and water of μl 300,000 c 17.05 ng 100 rxns 100 rxns Format 24 rxns ells
IPur e afterChIP STEP 1. STEP 4.DNAElution STEP 3.W STEP 2.DNAbinding cr Chr which ar DNA iselutedfr Pr magnet. DNA-bead c phosphate backboneofDNA. to bindthenegative Magnetic beadsacquire agar eluted fr downstr Purified DNAisr washed away Magnetic beadsf - Micr - Amplific - qPCR - Ne added. oss-linking andelution oteins andr omatin isdecr xt generationsequencing ose) whichar oarray Chr eam applic om beads(magneticor e disc ation ashes ompl omatin r . emaining buff ar w.ignd.o | www.diagenode.com om magneticbeads, ded. eady f ex or purific e disc os ation: isseparatedusinga ly slink char or any everse ar positivechar ed and ded. ation ar ged er ar
e e ge PAGE 45
STEP 4 DNA PURIFICATIONIPur e afterMeDIP STEP 4.DNAElution STEP 3.W STEP 2.DNAbinding STEP 1. which ar Magnetic beadsf (magnetic oragar DNA iselutedfr Remaining buff DNA iselutedfr to bindthenegative Magnetic Beadsacquire DNA-bead c phosphate backboneofDNA. magnet. added. downstr Purified DNAisr - Amplific - qPCR - Ne - Micr xt generationsequencing oarray DNA Elution eam applic e disc ation ashes ompl ar er iswashedaway om magneticbeads, om beads ded. eady f ex or purific ose). ation: isseparatedusinga ly char or any positivechar ation ar ged . e ge Innovating Epigenetic Solutions PAGE 46 STEP 5 LIBRARY PREPARATION Multiplexing from only 50pgofDNA 1 tube,2hours, 3steps! Preparation Kit MicroPlex Library DIAGENODE MicroPlex Library Preparation Kitv2x48(48indices) MicroPlex Library Preparation Kitv2x48(12indices) MicroPlex Library Preparation Kitv2x12(12indices) Product Replication Cleavable dsDNA template
Indexing options,easily automated Fewer PCRcycles: less bias Specifically optimized for DNA sample inputsfrom only 3 timesfaster thanTruSeq™ -only Simplified protocol: Stop CHROMATIN IMMUNOPRECIPITATION Stem-loop
Adaptor x ChIP’d DNA Primer A Nick excellent quality libraries that would be impossible to make using normal methods. Sequencing libraries and libraries well, material very starting Sequencing or performs kit The numbers methods. cell produce low genomes. normal and with large in using material studies even for especially make ChIP results of to future the libraries, in excellent amounts kit us impossible the give sequencing be use kit picogram will make with would we to MicroPlex start way that the from regularly efficient libraries We made most inputs. quality and low very quickest excellent the with is kit samples from MicroPlex Diagenode The + Single Library Preparation Library Repair Repair ChIP-seq tube,3-step protocol 50 pg
High FidelityAmplification Extension andCleavage Blunt-end Ligation Addition ofAdaptors DNA Repair 2 hours Ligate Ligate Dr. Morgan Sammons,LabofDr. Shelley Berger, University ofPennsylvania
Nick C05010014 C05010013 C05010012 + Cat. No. Amplification Clean-up Amplification Cl
ean-up Barcode Primer B x Stem-loop Adaptor Replication stop Cleavable sequencing (48 indices) (12 indices) (12 indices) Illumina Format 48 rxns 48 rxns 12 rxns ®
MicroPlex Kitwith: Combine the • ChIP-seq from only 10,000cells True MicroChIP kit Indexing reagents included in the kit the in reagentsIndexingincluded sequencing. for ready are libraries the purification, and After amplification protocol. 3-step simple and Illumina for libraries sequencing-ready into converted is dsDNA fragmented of 1: Figure sequencing onIllumina 4. Library purification and ligation, 3. Library amplification, procedure: 1. End-repair, 2. Adapter in asingle sequencinglane. allowmultiplexingthe samples12 of Figure 2:
® An input of 50 pg to 50 ng 50 to pg 50 of input An NGS platforms using a fast Library Preparation
® platform. PAGE 47 iDeal Library Preparation Kit
DNA sample inputs from 5 to 50 ng Validated with the IP-Star® Automated Platform Multiplexing capacity of up to 24 samples Only 3 hours from start to finish Ideal for use with our optimized MagMeDIP and iDeal ChIP-seq kits
Fragmented DNA Input PCR Enrichment P7 BC 5’ 5’ 3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’ 3’ 5’
5’ BC P7 End Repair, 5 Phosphorylation and dA-Talling 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’ 3’ 5’ 5’ 3’
P5 5’ Adaptor Ligation with optional iDeal Library Preparation 3’ 5’
5’ 3’ P5 T A 5’ U U 3’ 5’ A T 3’ 5’
5’ 3’ 3’ 5’ U Excision 5’ 3’ 5’ 3’ 3’ 5’
3’ 5’
Excision reagent 5’ 3’ 3’ 5’
5’ 3’ Clean up/Size selection 3’ 5’
5’ 3’ 3’ 5’ DNA P5 Primer Uracil P7 Primer 5’ 3’ 3’ 5’ Barcode (BC) Uracil excision reagent
T iDeal Library Adaptor
Clean up
A 1 2 B 1 2 High library yields 1500 5 ng of input DNA were used for each library prep. 700 7000 500 A. E. coli genomic DNA libraries. 1: Ladder; 2: Library prepared using 400 2000 1000 iDeal Library Preparation™ Kit. 300 600 500 400 B. FFPE sample. Human lung tumor genomic DNA 1: Library prepared 200 300 using the iDeal Library Preparation kit). 2: Input DNA (degraded FFPE 100 150 genomic DNA). 100 15 35
Product Cat. No. Format
iDeal Library Preparation™ kit x24 (incl. C05010020 24 rxns Index Primer Set 1)
iDeal Library Index Primer Set 2 C05010021 24 rxns
www.diagenode.com | DIAGENODE INNOVATING EPIGENETIC SOLUTIONS Diagenode, a world leader in the field of epigenetics develops and commercializes innovative instruments and reagents. Founded in 2003, Diagenode is a global company with headquarters in Liege (Belgium) and Denvile, NJ (USA). Products can also be purchased through our extensive dealer network.
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PUBLICATIONS On our website you can find links to hundreds of publications in which Diagenode’s products are described and reviewed. Visit our product pages or the publications page for more information: https://www.diagenode.com/publications. You can also ask our customer support ([email protected], [email protected]) to search our knowledge database to learn more about the technical aspects of our products and techniques, for information on independent reviews, or to find out how a product is used in a particular type of research.
TECHNICAL SUPPORT Please, do not hesitate to contact our customer support team if you have any questions about the design of your ChIP-seq experiment or the bioinformatics data analysis.
ASK THE Contact for Europe, Asia, Oceania and Africa: EXPERTS [email protected] Contact for North and South America: [email protected]
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