Chromatin Immunoprecipitation Protocol Research Articles

Chromatin Immunoprecipitation Sequencing (ChIP-seq) for Detecting Histone Modifications and Modifiers.

Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) is the most widely used method for analyzing genome-wide DNA-protein interactions. Because there is considerable variation in the modes and strengths of DNA-protein interactions, chromatin immunoprecipitation (ChIP) protocols have been diversified and optimized for different needs. Here, we describe protocols for detecting histone modifications and modifiers using various crosslinking and immunoprecipitation conditions. We provide a complete ChIP-seq workflow covering sample preparation, immunoprecipitation, next-generation sequencing (NGS) library preparation, and data analyses.

An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in peach reproductive tissues

BackgroundPerennial fruit trees display a growth behaviour characterized by annual cycling between growth and dormancy, with complex physiological features. Rosaceae fruit trees represent excellent models for studying not only the fruit growth/patterning but also the progression of the reproductive cycle depending upon the impact of climate conditions. Additionally, current developments in high‐throughput technologies have impacted Rosaceae tree research while investigating genome structure and function as well as (epi)genetic mechanisms involved in important developmental and environmental response processes during fruit tree growth. Among epigenetic mechanisms, chromatin remodelling mediated by histone modifications and other chromatin-related processes play a crucial role in gene modulation, controlling gene expression. Chromatin immunoprecipitation is an effective technique to investigate chromatin dynamics in plants. This technique is generally applied for studies on chromatin states and enrichment of post-transcriptional modifications (PTMs) in histone proteins.ResultsPeach is considered a model organism among climacteric fruits in the Rosaceae family for studies on bud formation, dormancy, and organ differentiation. In our work, we have primarily established specific protocols for chromatin extraction and immunoprecipitation in reproductive tissues of peach (Prunus persica). Subsequently, we focused our investigations on the role of two chromatin marks, namely the trimethylation of histone H3 at lysine in position 4 (H3K4me3) and trimethylation of histone H3 at lysine 27 (H3K27me3) in modulating specific gene expression. Bud dormancy and fruit growth were investigated in a nectarine genotype called Fantasia as our model system.ConclusionsWe present general strategies to optimize ChIP protocols for buds and mesocarp tissues of peach and analyze the correlation between gene expression and chromatin mark enrichment/depletion. The procedures proposed may be useful to evaluate any involvement of histone modifications in the regulation of gene expression during bud dormancy progression and core ripening in fruits.

Characterization of DNA-PK-Bound End Fragments Using GLASS-ChIP.

Endonucleolytic cleavage of DNA ends by the human Mre11-Rad50-Nbs1 (MRN) complex occurs in a manner that is promoted by DNA-dependent protein kinase (DNA-PK). A method is described to isolate DNA-PK-bound fragments released from chromatin in human cells using a modified Gentle Lysis and Size Selection chromatin immunoprecipitation (GLASS-ChIP) protocol. This method, combined with real-time PCR or next-generation sequencing, can identify sites of MRN endonucleolytic cutting adjacent to DNA-PK binding sites in human cells.

Transcription Factor Chromatin Immunoprecipitation in Endothelial Cells.

Interactions between DNA and proteins are crucial for the regulation of gene expression. Chromatin immunoprecipitation (ChIP) is a powerful technique that allows the study of specific protein-DNA interactions in cultured cells and fresh or fixed tissue. Chromatin is isolated and sheared, and antibodies against the protein(s) of interest are used to isolate specific protein-DNA complexes. Subsequent analysis by real-time polymerase chain reaction (qPCR) or next-generation sequencing (NGS) allows identification and quantification of the co-purified DNA fragments, and NGS also gives insight into the genomic binding sites of a protein. Here we describe a cross-linking ChIP (X-ChIP) protocol, based around the example of a myc-tagged Proline-Rich Homeodomain (PRH) protein expressed in human umbilical vein endothelial cells. We also describe how to analyse specific known or suspected binding sites using quantitative PCR as well as how to analyse genome-wide binding from ChIP sequencing data.

Characterization of DNA-PK-bound end fragments using GLASS-ChIP.

Endonucleolytic cleavage of DNA ends by the human Mre11-Rad50-Nbs1 (MRN) complex occurs in a manner that is promoted by DNA-dependent Protein Kinase (DNA-PK). A method is described to isolate DNA-PK-bound fragments released from chromatin in human cells using a modified Gentle Lysis and Size Selection chromatin immunoprecipitation (GLASS-ChIP) protocol. This method, combined with real-time PCR or next-generation sequencing, can identify sites of MRN endonucleolytic cutting adjacent to DNA-PK binding sites in human cells.

The Study of Protein-DNA Interactions in CD4+ T-Cells Using ChIPmentation.

Chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing (ChIP-seq) is an invaluable method to profile of enrichment of histone modifications and transcription factor binding sites across the genome. However, standard ChIP-seq protocols require large numbers of cells (>107) as starting material, which are often impossible to obtain for rare immune populations. Here we describe a streamlined ChIP protocol optimised for small cell numbers in conjunction with transposon-tagging mediated sequencing library preparation (ChIPmentation) which allows the analysis of samples of as low as 105 cells.

Profiling Protein-DNA Interactions by Chromatin Immunoprecipitation in Arabidopsis.

In plant cells, transcription factors play an important role in the regulation of gene expression, which eventually leads to the formation of complex phenotypes. Although chromatin immunoprecipitation (ChIP) involves a lengthy process that requires up to 4days to complete, it is a powerful technique to investigate the interactions between transcription factors and their target sequences in vivo. Here, we describe a detailed ChIP protocol, focusing on ChIP-qPCR, from material collection to data analyses. Moreover, we explain multiple checkpoints for the quality control of ChIP-qPCR data to ensure the success of this protocol. As this protocol is robust, it can be adapted to other plant materials and plant species, and it can be used for genome-wide profiling experiments, including ChIP-chip and ChIP-seq analyses. We believe that our ChIP-qPCR protocol facilitates research on the interactions between plant transcription factors and their target sequences in vivo.

An optimised chromatin immunoprecipitation (ChIP) method for starchy leaves of Nicotiana benthamiana to study histone modifications of an allotetraploid plant

All flowering plants have evolved through multiple rounds of polyploidy throughout the evolutionary process. Intergenomic interactions between subgenomes in polyploid plants are predicted to induce chromatin modifications such as histone modifications to regulate expression of gene homoeologs. Nicotiana benthamiana is an ancient allotetraploid plant with ecotypes collected from climatically diverse regions of Australia. Studying the chromatin landscape of this unique collection will likely shed light on the importance of chromatin modifications in gene regulation in polyploids as well its implications in adaptation of plants in environmentally diverse conditions. Generally, chromatin immunoprecipitation and high throughput DNA sequencing (ChIP-seq) is used to study chromatin modifications. However, due to the starchy nature of mature N. benthamiana leaves, previously published protocols were unsuitable. The higher amounts of starch in leaves that co-precipitated with nuclei hindered downstream processing of DNA. Here we present an optimised ChIP protocol for N. benthamiana leaves to facilitate comparison of chromatin modifications in two closely related ecotypes. Several steps of ChIP were optimised including tissue harvesting, nuclei isolation, nuclei storage, DNA shearing and DNA recovery. Commonly available antibodies targeting histone 3 lysine 4 trimethylation (H3K4me3) and histone 3 lysine 9 dimethylation (H3K9me2) histone modifications were used and success of ChIP was confirmed by PCR and next generation sequencing. Collectively, our optimised method is the first comprehensive ChIP method for mature starchy leaves of N. benthamiana to enable studies of chromatin landscape at the genome-wide scale.

Building a Robust Chromatin Immunoprecipitation Method with Substantially Improved Efficiency.

Chromatin immunoprecipitation (ChIP) is the gold-standard method for detection of interactions between proteins and chromatin and is a powerful tool for identification of epigenetic modifications. Although ChIP protocols for plant species have been developed, many specific features of plants, especially woody plants, still hinder the efficiency of immunoprecipitation, resulting in inefficient ChIP enrichment and an active demand for a highly efficient ChIP protocol. In this study, using birch (Betula platyphylla) and Arabidopsis (Arabidopsis thaliana) as the research materials, we identified five factors closely associated with ChIP efficiency, including crosslinking, concentration of chromatin using centrifugal filters, use of a different immunoprecipitation buffer, rescue of DNA with proteinase K, and use of Suc to increase immunoprecipitation efficiency. Optimization of any these factors can significantly improve ChIP efficiency. Considering these factors together, we developed a robust ChIP protocol that achieved a 14-fold improvement in ChIP enrichment for birch and a >6-fold improvement for Arabidopsis compared to the standard ChIP method. As this ChIP method works well in both birch and Arabidopsis, it should also be suitable for other woody and herbaceous species. In addition, this ChIP method enables detection of low-abundance transcription factor-DNA interactions and may extend the application of ChIP in the plant kingdom.

A native chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in strawberry fruits

BackgroundCovalent modifications of histones and histone variants have great influence on chromatin structure, which is involved in the transcriptional regulation of gene expression. Chromatin immunoprecipitation (ChIP) is a powerful tool for studying in vivo DNA-histone interactions. Strawberry is a model for Rosaceae and non-climacteric fruits, in which histone modifications have been implicated to affect fruit development and ripening. However, a validated ChIP method has not been reported in strawberry, probably due to its high levels of polysaccharides which affect the quality of prepared chromatin and the efficiency of immunoprecipitation.ResultsWe describe a native chromatin immunoprecipitation (N-ChIP) protocol suitable for strawberry by optimizing the parameters for nuclei isolation, chromatin extraction, DNA fragmentation and validation analysis using quantitative real-time PCR (qRT-PCR). The qRT-PCR results show that both the active mark H3K36me3 and the silent mark H3K9me2 are efficiently immunoprecipitated for the enriched regions. Compared to X-ChIP (cross-linked chromatin followed by immunoprecipitation), our optimized N-ChIP procedure has a higher signal-to-noise ratio and a lower background for both the active and the silent histone modifications. Furthermore, high-throughput sequencing following N-ChIP demonstrates that nearly 90% of the enriched H3K9/K14ac peaks are overlapped between biological replicates, indicating its remarkable consistency and reproducibility.ConclusionsAn N-ChIP method suitable for the fleshy fruit tissues of woodland strawberry Fragaria vesca is described in this study. The efficiency and reproducibility of our optimized N-ChIP protocol are validated by both qRT-PCR and high-throughput sequencing. We conclude that N-ChIP is a more suitable method for strawberry fruit tissues relative to X-ChIP, which could be combined with high-throughput sequencing to investigate the impact of histone modifications in strawberry and potentially in other fruits with high content of polysaccharides.

Measuring Histone Modifications in the Human Parasite Schistosoma mansoni.

DNA-binding proteins play critical roles in many major processes such as development and sexual biology of Schistosoma mansoni and are important for the pathogenesis of schistosomiasis. Chromatin immunoprecipitation (ChIP) experiments followed by sequencing (ChIP-seq) are useful to characterize the association of genomic regions with posttranslational chemical modifications of histone proteins. Challenges in the standard ChIP protocol have motivated recent enhancements in this approach, such as reducing the number of cells required and increasing the resolution. In this chapter, we describe the latest advances made by our group in the ChIP methods to improve the standard ChIP protocol to reduce the number of input cells required and to increase the resolution and robustness of ChIP in S. mansoni.

Chromatin Immunoprecipitation of Meiotically Expressed Proteins from Arabidopsis thaliana Flowers.

During meiosis recombination occurs between homologous chromosomes which can result in reciprocal exchanges of genetic information, called crossovers. Crossover rate is heterogeneous within the genome, with local regions having a significantly higher recombination rate relative to the genome average. These regions are termed hotspots and typically occur with widths of kilobases. Therefore, there is a need to profile recombination factors at a similar resolution during meiosis via techniques such as chromatin immunoprecipitation (ChIP). Here we describe a ChIP protocol, combined with high throughput sequencing (ChIP-seq) optimised for analysis of meiotically expressed proteins in Arabidopsis thaliana flowers. We provide methods to (1) isolate nuclei and prepare the chromatin for shearing, (2) immunoprecipitate DNA molecules cross-linked to a protein of interest, (3) to size-select and purify immunoprecipitated DNA molecules, and (4) to prepare DNA sequencing libraries suitable for high-throughput sequencing. Together, these methods allow the detection of binding sites for meiotic proteins in the Arabidopsis genome at high resolution, which will provide insights into relationships between meiotic chromosome organization, chromatin and recombination.

Going low to reach high: Small-scale ChIP-seq maps new terrain.

Chromatin immunoprecipitation (ChIP) enables mapping of specific histone modifications or chromatin-associated factors in the genome and represents a powerful tool in the study of chromatin and genome regulation. Importantly, recent technological advances that couple ChIP with whole-genome high-throughput sequencing (ChIP-seq) now allow the mapping of chromatin factors throughout the genome. However, the requirement for large amounts of ChIP-seq input material has long made it challenging to assess chromatin profiles of cell types only available in limited numbers. For many cell types, it is not feasible to reach high numbers when collecting them as homogeneous cell populations in vivo. Nonetheless, it is an advantage to work with pure cell populations to reach robust biological conclusions. Here, we review (a) how ChIP protocols have been scaled down for use with as little as a few hundred cells; (b) which considerations to be aware of when preparing small-scale ChIP-seq and analyzing data; and (c) the potential of small-scale ChIP-seq datasets for elucidating chromatin dynamics in various biological systems, including some examples such as oocyte maturation and preimplantation embryo development. This article is categorized under: Laboratory Methods and Technologies > Genetic/Genomic Methods Developmental Biology > Developmental Processes in Health and Disease Biological Mechanisms > Cell Fates.

Rapid method for chromatin immunoprecipitation (ChIP) assay in a dimorphic fungus, Candida albicans.

A chromatin immunoprecipitation (ChIP) assay is a method to identify how much a protein of interest binds to the DNA region. This method is indispensable to study the mechanisms of how the transcription factors or chromatin modifications regulate the gene expression. Candida albicans is a dimorphic pathogenic fungus, which can change its morphology very rapidly from yeast to hypha in response to the environmental signal. The morphological change of C. albicans is one of the critical factors for its virulence. Therefore, it is necessary to understand how to regulate the expression of genes for C. albicans to change its morphology. One of the essential methods for us to understand this regulation is a ChIP assay. There have been many efforts to optimize the protocol to lower the background signal and to analyze the results accurately because a ChIP assay can provide very different results even with slight differences in the experimental procedure. We have optimized the rapid and efficient ChIP protocol so that it could be applied equally for both yeast and hyphal forms of C. albicans. Our method in this protocol is also comparatively rapid to the method widely used. In this protocol, we described our rapid method for the ChIP assay in C. albicans in detail.

Effects of low-temperature stress on histone modification in ZF4 cells

PDF HTML阅读 XML下载 导出引用 引用提醒 低温压力对ZF4细胞组蛋白修饰的影响 DOI: 作者: 作者单位: 1. 水产种质资源发掘与利用教育部重点实验室, 上海海洋大学, 上海 201306;2. 水产科学国家级实验教学示范中心, 上海海洋大学, 上海 201306;3. 海洋生物科学国际联合研究中心, 中国科学技术部, 上海海洋大学, 上海 201306 作者简介: 姜蓬垒(1988-),男,博士研究生,研究方向为表观遗传学.E-mail:770163089@qq.com 通讯作者: 中图分类号: S917 基金项目: 国家自然科学基金项目（31372516，81770165）；上海市自然科学基金项目（13ZR1419500）；上海市教育委员会"东方学者"计划支持项目. Effects of low-temperature stress on histone modification in ZF4 cells Author: Affiliation: 1. Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education;Shanghai Ocean University, Shanghai 201306, China;2. National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China;3. International Research Center for Marine Biosciences at Shanghai Ocean University, Ministry of Science and Tech-nology, Shanghai 201306, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:为探索低温压力对斑马鱼细胞组蛋白修饰的影响，建立并优化使用斑马鱼（）成纤维细胞ZF4进行染色质免疫共沉淀（chromatin immunoprecipitation，ChIP）的条件。本研究分别优化了ChIP过程中裂解细胞所使用的NP-40浓度、超声破碎的时间，确定了最佳NP-40浓度为0.2%，超声时间为20 min。利用针对-actin启动子区域的引物，通过常规PCR初步验证ChIP实验是否成功。琼脂糖凝胶电泳结果显示，IgG组常规PCR产物基本无条带，H3K27me3组条带较暗，H3K4me3和H3K27ac组有较亮条带，初步说明实验可靠。使用正常培养（28℃）与长期低温驯化（18℃，30 d）的斑马鱼成纤维细胞ZF4作为实验材料，通过优化之后的ChIP技术进行实验。分别使用qPCR和ChIP-qPCR检测低温驯化对肿瘤坏死因子b（tumor necrosis factor b，基因经过低温驯化后上调，而ChIP-qPCR结果显示，基因启动子区域的H3K4me3、H3K27ac水平来调控基因的表达。本研究建立并优化的ChIP实验条件可以用来研究ZF4细胞组蛋白修饰变化，为下一步探索低温压力对斑马鱼细胞全基因组组蛋白修饰的影响奠定基础。 Abstract:Environmental stresses can regulate gene expression patterns by modulating histone activity. However, the effect of low-temperature stress on whole-genome histone modification in fish has not been reported. To explore the effect of low-temperature stress on histone modification in zebrafish () cells, the experimental conditions for chromatin immunoprecipitation (ChIP) were established and optimized. The optimum NP-40 concentration for lysing cells was 0.2%. The optimal time of sonication with a Covaris S220 to break the chromatin was 20 min. Primers targeting the -actin promoter region were used to perform routine PCR to verify the preliminary ChIP experiment results. Optimized experimental conditions resulted in a very low background, with nearly no IgG band in the agarose gel. Moreover, the lack of H3K27me3 in the -actin promoter region, accompanied by enriched H3K4me3 and H3K27ac, was observed as predicted. Zebrafish embryonic ZF4 fibroblasts cultured under normal condition (28℃) and cold-acclimated ZF4 cells under long-term cold exposure (18℃ for 30 days) were evaluated with the optimized ChIP protocol. The effect of cold acclimation on the expression of tumor necrosis factor tnfb promoter was detected by qPCR and ChIP-qPCR, respectively. The qPCR and ChIP-qPCR results showed that was upregulated after cold acclimation and that H3K4me3 and H3K27ac were enriched in the promoter region of , while enrichment of H3K27me3 showed no significant change. This suggests that cold pressure regulates expression by affecting H3K4me3 and H3K27ac levels in the promoter region of . In conclusion, the established and optimized ChIP method can be used to study histone modification in ZF4 cells and provides a foundation for further analysis of the effect of low-temperature stress on whole-genome histone modification in zebrafish cells. 参考文献 相似文献 引证文献

Genome-wide identification of RETINOBLASTOMA RELATED 1 binding sites in Arabidopsis reveals novel DNA damage regulators.

Retinoblastoma (pRb) is a multifunctional regulator, which was likely present in the last common ancestor of all eukaryotes. The Arabidopsis pRb homolog RETINOBLASTOMA RELATED 1 (RBR1), similar to its animal counterparts, controls not only cell proliferation but is also implicated in developmental decisions, stress responses and maintenance of genome integrity. Although most functions of pRb-type proteins involve chromatin association, a genome-wide understanding of RBR1 binding sites in Arabidopsis is still missing. Here, we present a plant chromatin immunoprecipitation protocol optimized for genome-wide studies of indirectly DNA-bound proteins like RBR1. Our analysis revealed binding of Arabidopsis RBR1 to approximately 1000 genes and roughly 500 transposable elements, preferentially MITES. The RBR1-decorated genes broadly overlap with previously identified targets of two major transcription factors controlling the cell cycle, i.e. E2F and MYB3R3 and represent a robust inventory of RBR1-targets in dividing cells. Consistently, enriched motifs in the RBR1-marked domains include sequences related to the E2F consensus site and the MSA-core element bound by MYB3R transcription factors. Following up a key role of RBR1 in DNA damage response, we performed a meta-analysis combining the information about the RBR1-binding sites with genome-wide expression studies under DNA stress. As a result, we present the identification and mutant characterization of three novel genes required for growth upon genotoxic stress.

Canonical BMP signaling in tubular cells mediates recovery after acute kidney injury

Bone morphogenetic protein (BMP) signaling has been shown to modulate the development of renal fibrosis in animal models of kidney injury, but the downstream mediators are incompletely understood. In wild-type mice, canonical BMP signaling mediated by SMAD1/5/8 transcription factors was constitutively active in healthy renal tubules, transiently down-regulated after ischemia reperfusion injury (IRI), and reactivated during successful tubular regeneration. We then induced IRI in mice with a tubular-specific BMP receptor 1A (BMPR1A) deletion. These mice failed to reactivate SMAD1/5/8 signaling in the post-ischemic phase and developed renal fibrosis after injury. Using unbiased genomic analyses, we identified three genes encoding inhibitor of DNA-binding (ID) proteins (Id1, Id2, and Id4) as key targets of BMPR1A-SMAD1/5/8 signaling. BMPR1A-deficient mice failed to re-induce these targets following IRI. Instead, BMPR1A-deficiency resulted in activation of pro-fibrotic signaling proteins that are normally repressed by ID proteins, namely, p38 mitogen-activated protein kinase and cell cycle inhibitor p27. These data indicate that the post-ischemic activation of canonical BMP signaling acts endogenously to repress pro-fibrotic signaling in tubular cells and may help to prevent the progression of acute kidney injury to chronic kidney disease.

Using ChIP-Based Approaches to Characterize FOXO Recruitment to its Target Promoters.

Chromatin immunoprecipitation (ChIP) coupled to quantitative real-time PCR (ChIP-qPCR) or Next-Generation Sequencing (ChIP-seq) enables us to study the dynamics of chromatin recruitment of transcription factors (TFs). The popular model system Caenorhabditis elegans has provided us with fundamental understanding of the role of Insulin/IGF-1-like signaling (IIS) in metabolism and aging. The FOXO TF DAF-16 is the major output of the pathway that regulates most of the phenotypes associated with the IIS pathway. Here, we describe a ChIP protocol to study FOXO recruitment dynamics in whole C. elegans extracts. We discuss detailed practical procedures, including optimization, growth, harvesting, formaldehyde fixation, sonication of worms, TF immunoprecipitation for further downstream processing using qPCR as well as NGS for the analysis of FOXO-bound DNA.

Optimization of ChIP protocol for investigation of canonical Wnt target genes expression in new born heart

Aim. In present work we have focused on chromatinimmunoprecipitation (ChIP) protocol with next qPCR optimization for analysis of canonical Wnt target genes regulation by b‑catenin and plakoglobin in new born heart. Methods. In study we have used the new born hearts tissue at the age of 3 days after birth (P3). We have analyzed hearts of control mice and mice with heterozygous knockout of β-catenin. We have proceeded ChIP protocol based on standard protocols in our modifications. ChIP results validated with qPCR. Results. We have adopted ChIP protocol for investigation of signalling function of β-catenin and plakoglobin as well as for identification of β-catenin and plakoglobin specific binding genomic region in new born heart. Conclusions. We have shown that plakoglobin is able to compensate the signaling function of β-catenin and binds with similar genomic region under β-catenin haploinsufficiency conditions.
 Keywords: β-catenin, plakoglobin, Wnt signaling, gene expression, heart, chromatinimmunoprecipitation, ChIP.

Chromatin Immunoprecipitation for Chromatin Interaction Analysis Using Paired-End-Tag (ChIA-PET) Sequencing in Tadpole Tissues.

Proper gene expression involves communication between the regulatory elements and promoters of genes. Because regulatory elements can be located over a large range of genomic distances (from as close as a few hundred bp to as much as several Mb away), contact and communication between regulators and the core transcriptional machinery at promoters are mediated through DNA looping. Today, chromosome conformation capture (3C)-based methods efficiently probe chromosome folding in the nucleus and thus provide a molecular description of physical proximity between enhancer(s) and their target promoter(s). One such method, chromatin interaction analysis using paired-end-tag (ChIA-PET) sequencing, is a leading high-throughput method for detection of genome wide chromatin interactions. Briefly, the method involves cross-linkage of chromatin (-DNA) fibers in cells in situ, fragmentation of the fixed chromatin-DNA complexes by sonication, followed by enrichment of the chromatin complexes with a dedicated antibody through the process of immunoprecipitation (IP). Next, application of the ChIA-PET protocol followed by deep sequencing and mapping of reads to the reference genome reveals both binding sites and remote chromatin interactions mediated by the protein factors of interest. The method detailed here focuses on ChIP sample preparation and can be completed in ∼5 d. The ChIA-PET method is detailed in an associated protocol. Because not all chromatin immunoprecipitation protocols are suitable for ChIA-PET, it is important to strictly follow this procedure before performing the ChIA-PET protocol.