Chromatin Immunoprecipitation Protocol Research Articles

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo.

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions <em>In Vivo</em>

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

From the laboratory to the field: assaying histone methylation at <i>FLOWERING LOCUS C</i> in naturally growing <i>Arabidopsis halleri</i>

Gene regulatory mechanisms are often defined in studies performed in the laboratory but are seldom validated for natural habitat conditions, i.e., in natura. Vernalization, the promotion of flowering by winter cold, is a prominent naturally occurring phenomenon, so far best characterized using artificial warm and cold treatments. The floral inhibitor FLOWERING LOCUS C (FLC) gene of Arabidopsis thaliana has been identified as the central regulator of vernalization. FLC shows an idiosyncratic pattern of histone modification at different stages of cold exposure, believed to regulate transcriptional responses of FLC. Chromatin modifications, including H3K4me3 and H3K27me3, are routinely quantified using chromatin immunoprecipitation (ChIP), standardized for laboratory samples. In this report, we modified a ChIP protocol to make it suitable for analysis of field samples. We first validated candidate normalization control genes at two stages of cold exposure in the laboratory and two seasons in the field, also taking into account nucleosome density. We further describe experimental conditions for performing sampling and sample preservation in the field and demonstrate that these conditions give robust results, comparable with those from laboratory samples. The ChIP protocol incorporating these modifications, "Field ChIP", was used to initiate in natura chromatin analysis of AhgFLC, an FLC orthologue in A. halleri, of which a natural population is already under investigation. Here, we report results on levels of H3K4me3 and H3K27me3 at three representative regions of AhgFLC in controlled cold and field samples, before and during cold exposure. We directly compared the results in the field with those from laboratory samples. These data revealed largely similar trends in histone modification dynamics between laboratory and field samples at AhgFLC, but also identified some possible differences. The Field ChIP method described here will facilitate comprehensive chromatin analysis of AhgFLC in the future to contribute to our understanding of gene regulation in fluctuating natural environments.

Mapping Recombination Initiation Sites Using Chromatin Immunoprecipitation.

Genome-wide maps of recombination sites provide valuable information not only on the recombination pathway itself but also facilitate the understanding of genome dynamics and evolution. Here, we describe a chromatin immunoprecipitation (ChIP) protocol to map the sites of recombination initiation in plants with maize used as an example. ChIP is a method that allows identification of chromosomal sites occupied by specific proteins. Our protocol utilizes RAD51, a protein involved in repair of double-strand breaks (DSBs) that initiate meiotic recombination, to identify DSB formation hotspots. Chromatin is extracted from meiotic flowers, sheared and enriched in fragments bound to RAD51. Genomic location of the protein is then identified by next-generation sequencing. This protocol can also be used in other species of plants, animals, and fungi.

Mapping Protein-DNA Interactions Using ChIP-exo and Illumina-Based Sequencing.

Chromatin immunoprecipitation (ChIP) provides a means of enriching DNA associated with transcription factors, histone modifications, and indeed any other proteins for which suitably characterized antibodies are available. Over the years, sequence detection has progressed from quantitative real-time PCR and Southern blotting to microarrays (ChIP-chip) and now high-throughput sequencing (ChIP-seq). This progression has vastly increased the sequence coverage and data volumes generated. This in turn has enabled informaticians to predict the identity of multi-protein complexes on DNA based on the overrepresentation of sequence motifs in DNA enriched by ChIP with a single antibody against a single protein. In the course of the development of high-throughput sequencing, little has changed in the ChIP methodology until recently. In the last three years, a number of modifications have been made to the ChIP protocol with the goal of enhancing the sensitivity of the method and further reducing the levels of nonspecific background sequences in ChIPped samples. In this chapter, we provide a brief commentary on these methodological changes and describe a detailed ChIP-exo method able to generate narrower peaks and greater peak coverage from ChIPped material.

Chromatin Immunoprecipitation (ChIP) Assay in Candida albicans.

Chromatin immunoprecipitation (ChIP) is a widely used technique which can determine the in vivo association of a specific protein on a particular DNA locus in the genome. In this method cross-linked chromatin is sheared and immunoprecipitated with antibodies raised against a target protein of interest. The end result of this process is the enrichment of DNA fragments associated with the desired protein. Thus, interactions between proteins and genomic loci in cellular context can be determined by this technique. Here, we are describing a ChIP protocol that is optimized for Candida albicans. The protocol requires 4-5 days for completion of the assay and has been used to produce robust ChIP results for diverse proteins in this organism and its related species including Candida dubliniensis and Candida tropicalis.

Chromatin Immunoprecipitation for Detecting Epigenetic Marks on Plant Nucleosomes.

Due to high resolution and reproducibility, chromatin immunoprecipitation (ChIP) has been used as a standard tool to investigate epigenetic marks including modified histones and specific histone variants (e.g., centromere-specific histone H3, CENH3) in this decade. Here, I describe a sensitive and low-background ChIP protocol for a wide range of plant species.

ChIP bias as a function of cross-linking time.

The chromatin immunoprecipitation (ChIP) assay is widely used to capture interactions between chromatin and regulatory proteins in vivo. Formaldehyde cross-linking of DNA and proteins is a critical step required to trap their interactions inside the cells before immunoprecipitation and analysis. Yet insufficient attention has been given to variables that might give rise to artifacts in this procedure, such as the duration of cross-linking. We analyzed the dependence of the ChIP signal on the duration of formaldehyde cross-linking time for two proteins: DNA topoisomerase 1 (Top1) that is functionally associated with the double helix in vivo, especially with active chromatin, and green fluorescent protein (GFP) that has no known bona fide interactions with DNA. With short time of formaldehyde fixation, only Top1 immunoprecipation efficiently recovered DNA from active promoters, whereas prolonged fixation augmented non-specific recovery of GFP dramatizing the need to optimize ChIP protocols to minimize the time of cross-linking, especially for abundant nuclear proteins. Thus, ChIP is a powerful approach to study the localization of protein on the genome when care is taken to manage potential artifacts.

Dynamic changes in histone modifications precede de novo DNA methylation in oocytes.

Erasure and subsequent reinstatement of DNA methylation in the germline, especially at imprinted CpG islands (CGIs), is crucial to embryogenesis in mammals. The mechanisms underlying DNA methylation establishment remain poorly understood, but a number of post-translational modifications of histones are implicated in antagonizing or recruiting the de novo DNA methylation complex. In mouse oogenesis, DNA methylation establishment occurs on a largely unmethylated genome and in nondividing cells, making it a highly informative model for examining how histone modifications can shape the DNA methylome. Using a chromatin immunoprecipitation (ChIP) and genome-wide sequencing (ChIP-seq) protocol optimized for low cell numbers and novel techniques for isolating primary and growing oocytes, profiles were generated for histone modifications implicated in promoting or inhibiting DNA methylation. CGIs destined for DNA methylation show reduced protective H3K4 dimethylation (H3K4me2) and trimethylation (H3K4me3) in both primary and growing oocytes, while permissive H3K36me3 increases specifically at these CGIs in growing oocytes. Methylome profiling of oocytes deficient in H3K4 demethylase KDM1A or KDM1B indicated that removal of H3K4 methylation is necessary for proper methylation establishment at CGIs. This work represents the first systematic study performing ChIP-seq in oocytes and shows that histone remodeling in the mammalian oocyte helps direct de novo DNA methylation events.

Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins.

We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols.

MOBE-ChIP: a large-scale chromatin immunoprecipitation assay for cell type-specific studies.

Cell type-specific transcriptional regulators play critical roles in the generation and maintenance of multicellularity. As they are often expressed at low levels, in vivo DNA-binding studies of these regulators by standard chromatin immunoprecipitation (ChIP) assays are technically challenging. We describe here an optimized ChIP protocol termed Maximized Objects for Better Enrichment (MOBE)-ChIP, which enhances the sensitivity of ChIP assays for detecting cell type-specific signals. The protocol, which is based on the disproportional increase of target signals over background at higher scales, uses substantially greater volume of starting materials than conventional ChIPs to achieve high signal enrichment. This technique can capture weak binding events that are ambiguous in standard ChIP assays, and is useful both in gene-specific and whole-genome analysis. This protocol has been optimized for Arabidopsis, but should be applicable to other model systems with minor modifications. The full procedure can be completed within 3 days.

Chromatin analyses of Zymoseptoria tritici: Methods for chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq)

The presence or absence of specific transcription factors, chromatin remodeling machineries, chromatin modification enzymes, post-translational histone modifications and histone variants all play crucial roles in the regulation of pathogenicity genes. Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) provides an important tool to study genome-wide protein–DNA interactions to help understand gene regulation in the context of native chromatin. ChIP-seq is a convenient in vivo technique to identify, map and characterize occupancy of specific DNA fragments with proteins against which specific antibodies exist or which can be epitope-tagged in vivo. We optimized existing ChIP protocols for use in the wheat pathogen Zymoseptoria tritici and closely related sister species. Here, we provide a detailed method, underscoring which aspects of the technique are organism-specific. Library preparation for Illumina sequencing is described, as this is currently the most widely used ChIP-seq method. One approach for the analysis and visualization of representative sequence is described; improved tools for these analyses are constantly being developed. Using ChIP-seq with antibodies against H3K4me2, which is considered a mark for euchromatin or H3K9me3 and H3K27me3, which are considered marks for heterochromatin, the overall distribution of euchromatin and heterochromatin in the genome of Z. tritici can be determined. Our ChIP-seq protocol was also successfully applied to Z. tritici strains with high levels of melanization or aberrant colony morphology, and to different species of the genus (Z. ardabiliae and Z. pseudotritici), suggesting that our technique is robust. The methods described here provide a powerful framework to study new aspects of chromatin biology and gene regulation in this prominent wheat pathogen.

Micro chromatin immunoprecipitation (μChIP) from early mammalian embryos.

Chromatin immunoprecipitation (ChIP) is a powerful method for mapping protein-DNA interactions in vivo. Genomic localization of histone modifications, transcription factors, and other regulatory proteins can be revealed by ChIP. However, conventional ChIP protocols require the use of large numbers of cells, which prevents the application of ChIP to rare cell types. We have developed ChIP assays suited for the immunoprecipitation of histone proteins or transcription factors from small cell numbers. Here we describe a rapid, yet sensitive micro (μ)ChIP protocol producing high signal to noise ratio output, suitable for as few as 100 cells. This chapter provides a detailed protocol for μChIP from early mammalian embryos, also suitable for any sample of limited numbers of cells. Minor modifications of this optimized high signal to noise ChIP protocol make it a reliable tool for the use with any cell number (100-10(7)).

A robust chromatin immunoprecipitation protocol for studying transcription factor-DNA interactions and histone modifications in wood-forming tissue.

Woody cells and tissues are recalcitrant to standard chromatin immunoprecipitation (ChIP) procedures. However, we recently successfully implemented ChIP in wood-forming tissue of the model woody plant Populus trichocarpa. Here we provide the detailed ChIP protocol optimized for wood-forming tissue that we used in those studies. By using stem-differentiating xylem (SDX; a wood-forming tissue), we identified all steps that were ineffective in standard ChIP protocols and systematically modified them to develop and optimize a robust ChIP protocol. The protocol includes tissue collection, cross-linking, nuclear isolation, chromatin extraction, DNA fragmentation, immunoprecipitation, DNA purification and sequence analysis. The protocol takes 2.5 d to complete and allows a robust 8-10-fold enrichment of transcription factor (TF)-bound genomic fragments (~150 ng/g of SDX) over nonspecific DNAs. The enriched DNAs are of high quality and can be used for subsequent PCR and DNA-seq analyses. We used this protocol to identify genome-wide specific TF-DNA interactions during wood formation and histone modifications associated with regulation of wood formation. Our protocol, which may be suitable for many tissue types, is so far the only working ChIP system for wood-forming tissue.

Genome-wide profiling of histone 3 lysine 36 trimethylation in clear cell renal cell carcinoma.

464 Background: Although the von Hippel-Lindau (VHL) tumor suppressor gene is mutated in 60% of ccRCC, deletion of VHL in mice is insufficient for tumorigenesis. Sequencing of ccRCC tumors identified mutations in SETD2, a histone H3 lysine 36 (H3K36) trimethyltransferase. We hypothesize that loss of SETD2 methyltransferase activity alters the genome wide pattern of H3K36 trimethylation (H3K36me3) in ccRCC, and contributes to the cancer phenotype. Methods: To generate a genome-wide profile of H3K36me3 in frozen nephrectomy samples and RCC cell lines, we optimized a chromatin immunoprecipitation (ChIP) protocol for the isolation of DNA associated with H3K36me3. H3K36me3 is associated with open chromatin and an H3K36me3-specific antibody was used for immunoprecipitation of endogenous H3K36me3-bound DNA. ChIP PCR primers were optimized for active genes, such as actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a “gene desert” on chromosome 12 (negative control). ChIP libraries were then generated from 3 paired uninvolved kidney and RCC and 2 RCC cell lines. In order to identify H3K36Me3 upregulated regions in uninvolved kidney and RCC, reads from the ChIP sequencing were mapped to the human genome using Burrows-Wheeler Aligner and SICER algorithms. Results: Using ChIP PCR, we found that active genomic regions were enriched 15-30 fold over the negative controls indicating that the quality and yield of immunoprecipitated DNA/chromatin complexes from frozen tissue was sufficient for ChIP sequencing. A preliminary ChIP sequencing analysis of RCC cell lines and frozen ccRCC tissue indicates that H3K36me3 enriched DNA sequences were mapped to exons (31.3%) compared to introns (13.5%, p&lt;0.001), consistent with the role of H3K36me3 in transcription. Conclusions: Genomic regions enriched for H3K36Me3 binding were identified from patient-derived tissue and RCC cell lines. Current efforts are focused on comparing the H3K36me3 profiles between matched tumor and uninvolved kidney ChIP libraries to generate a genome wide map of dysregulated H3K36me3 modifications.

ChIP-seq analysis of histone modifications at the core of the Arabidopsis circadian clock.

Over the past years, chromatin modification has emerged as a key regulator of gene expression. A very useful method for chromatin analysis is chromatin immunoprecipitation (ChIP), which allows the quantification and localization of specific histone modifications. The basic steps of the ChIP protocol include cross-linking of histones and DNA, chromatin isolation, shearing the DNA into smaller fragments, immunoprecipitation with specific antibodies, and enrichment analysis by several methods including real-time quantitative PCR (ChIP-qPCR), microarray hybridization (ChIP-chip), or sequencing (ChIP-seq). Here, we describe how to use ChIP-qPCR to analyze histone modifications at the core of the Arabidopsis thaliana circadian clock. We also briefly discuss a number of protocol adjustments to be considered in ChIP-seq experiments.

A nuclear role of FKBP51?

A nuclear role of FKBP51? FKBP51 is an immunophilin and chaperon that plays an important role in hormone receptor control. A single nucleotide polymorphism (SNP) in FKBP51 has been associated with different courses of disease in depression. Therefor it is considered a potential drug-target. With its tetratricopeptide repeat and cis-trans-propyl-isomerase domains it has been shown to interact with mature corticoid receptor hetero-complexes and protein kinases like Akt. While all the known functions of FKBP51 are performed in the cytoplasm, biochemical fractionation and confocal microscopy have shown that FKBP51 is not only a cytosolic protein, but is also found in the nucleus. We also observed this phenomenon in our lab and decided to map FKBP51 to the genome. To this end, we are currently optimizing a chromatin-immunoprecipitation (ChIP) procedure. We expect that FKBP51 is not bound directly to the DNA, but is associated to it through a mediator protein; this requires careful optimization of the fixation parameters. After successful establishment of the ChIP protocol we plan to map the DNA binding sites using next-generation sequencing. Revealing possible chromatin functions of FKBP51 could shed light on its role in depression; moreover, a comprehensive knowledge of the chromatin sites of FKBP51 may lead to more targeted drug development that addresses some, but not all functions of FKBP51.

A chromatin immunoprecipitation (ChIP) protocol for use in whole human adipose tissue

Chromatin immunoprecipitation (ChIP) has become a central method when studying in vivo protein-DNA interactions, with the major challenge being the hope to capture "authentic" interactions. While ChIP protocols have been optimized for use with specific cell types and tissues including adipose tissue-derived cells, a working ChIP protocol addressing the challenges imposed by fresh whole human adipose tissue has not been described. Utilizing human paired omental and subcutaneous adipose tissue obtained during elective abdominal surgeries, we have carefully identified and optimized individual steps in the ChIP protocol employed directly on fresh tissue fragments. We describe a complete working protocol for using ChIP on whole adipose tissue fragments. Specific steps required adaptation of the ChIP protocol to human whole adipose tissue. In particular, a cross-linking step was performed directly on fresh small tissue fragments. Nuclei were isolated before releasing chromatin, allowing better management of fat content; a sonication protocol to obtain fragmented chromatin was optimized. We also demonstrate the high sensitivity of immunoprecipitated chromatin from adipose tissue to freezing. In conclusion, we describe the development of a ChIP protocol optimized for use in studying whole human adipose tissue, providing solutions for the unique challenges imposed by this tissue. Unraveling protein-DNA interaction in whole human adipose tissue will likely contribute to elucidating molecular pathways contributing to common human diseases such as obesity and type 2 diabetes.

Abstract A58: Systematic optimization of key parameters involved in the preparation of chromatin and the chromatin immunoprecipitation (ChIP) workflow

Abstract The ChIP protocol is a fundamental tool that has been used to gain insight into the impact of epigenetic phenomenon and their effect on cancer development and progression. Despite the abundance of research tools that support ChIP, very little innovation has occurred around the ChIP method itself to make the process more reproducible and reliable. Issues surrounding antibody specificity and performance, lack of reliable high-throughput options for limited amounts of chromatin, and accepted criteria to make determinations on the quality of chromatin suitable for ChIP are just some of the challenges faced when performing this method. Improvements in the ChIP process would enable more reliable high resolution genomic mapping of chromatin events that are critical to unambiguously dissect epigenetic regulatory mechanisms and their influence on gene expression. To address these issues, we have engaged in an iterative process of examining multiple parameters that influence ChIP data. For chromatin fragmentation, we prepared chromatin on a variety of devices and utilized various buffer systems to develop an approach to optimize this process. To allow rapid and evaluation of histone antibody specificity, we have produced tools that allow researchers to evaluate specificity utilizing a simple dot blot western blotting approach. In addition, we have developed low-cell protocol that simplifies the ChIP process, reduces variability and significantly improve signal-to-noise ratios. This protocol has been demonstrated to work in both a low throughput version as well as a high-throughput version that is amenable to automation. Our approach is well suited to those with limited experience in ChIP, but is also sufficiently robust and flexible to enable ChIP from either abundant or limited amounts of chromatin derived from cells or tissues. Using this systematic approach, we present data comparing the impact of different sonication protocols on ChIP, analysis of user-to-user variation, as well as approaches to optimize and control for factors that introduce variability in ChIP. Citation Format: Tracy Cooke, Zirong Li, Kan Saito, Michael R. Sturges, Konstantin Taganov, Bhaskar Thyagarajan, John M. Rosenfeld, Alejandra Solache. Systematic optimization of key parameters involved in the preparation of chromatin and the chromatin immunoprecipitation (ChIP) workflow. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A58.

Abstract B58: Implementation of a synthetic reagent to mimic chromatin provides a chromatin immunoprecipitation quantitative control

Abstract Chromatin immunoprecipitation (ChIP) is an analytical method used to investigate interaction of proteins with specific genomic DNA regions in vivo to provide a better understanding of mechanisms of gene regulation, DNA replication and DNA repair. However, variations in the efficiency of immunoprecipitation, background signals from carryover in immunoprecipitation, variability between chromatin preps, and loss of material during immunoprecipitation and purification of the ChIP DNA are sources of variability that restrict the use of ChIP as a quantitative tool. To address these limitations, we have developed a simple spike-in control that enables labs to generate results that are more consistent and more quantitative. The approach we have developed uses a synthetic peptide-DNA reagent that is spiked into chromatin immunoprecipitation reactions. Because this reagent mimics the behavior of cross-linked chromatin and can be quantitatively recovered by immunoprecipitation, it can be readily incorporated in to virtually any ChIP protocol. To utilize this internal control, a fixed amount of the synthetic peptide-DNA complex is spiked into the chromatin and co-ChIPed simultaneously with the antibody of interest. The recovered synthetic DNA is then used to normalize the results obtained with the antibody of interest. This spike-in control can be designed using a peptide specific to the ChIP antibody of interest and recovered using a single antibody. Alternatively, a universal spike-in control can be designed using a peptide that is recognized by an epitope tag antibody. This universal spike-in control and epitope tagged antibody are used along with the antibody of interest in a standard ChIP reaction. Our data show that the inclusion of our internal control significantly reduces variability between ChIP assays, thus increasing the accuracy of the ChIP data. Because of the universal feature, this method can also be used to compare ChIP data between different antibodies or to normalize data variations that often result between users and multiple experiments. Furthermore, experiments are underway to confirm that such a spike in control can be incorporated upstream of chromatin preparation, and is capable of being adapted for NGS library construction to aid in normalization between chromatin NGS experiments. Citation Format: John M. Rosenfeld, Zirong Li, Konstantin Taganov, Tracy Cooke, Bhaskar Thyagarajan, Alejandra Solache. Implementation of a synthetic reagent to mimic chromatin provides a chromatin immunoprecipitation quantitative control. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B58.