aChIP is an efficient and sensitive ChIP-seq technique for economically important plant organs.

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is crucial for profiling histone modifications and transcription factor binding throughout the genome. However, its application in economically important plant organs (EIPOs) such as seeds, fruits and flowers is challenging due to their sturdy cell walls and complex constituents. Here we present advanced ChIP (aChIP), an optimized method that efficiently isolates chromatin from plant tissues while simultaneously removing cell walls and cellular constituents. aChIP precisely profiles histone modifications in all 14 tested EIPOs and identifies transcription factor and chromatin-modifying enzyme binding sites. In addition, aChIP enhances ChIP efficiency, revealing numerous novel modified sites compared with previous methods in vegetative tissues. aChIP reveals the histone modification landscape for rapeseed dry seeds, highlighting the intricate roles of chromatin dynamics during seed dormancy and germination. Altogether, aChIP is a powerful, efficient and sensitive approach for comprehensive chromatin profiling in virtually all plant tissues, especially in EIPOs.

Efficient Enrichment of Synchronized Mouse Spermatocytes Suitable for Genome-Wide Analysis.

Chromatin undergoes extensive remodeling during meiosis, leading to specific patterns of gene expression and chromosome organization, which ultimately controls fundamental meiotic processes such as recombination and homologous chromosome associations. Recent game-changing advances have been made by analysis of chromatin binding sites of meiotic specific proteins genome-wide in mouse spermatocytes. However, further progress is still highly dependent on the reliable isolation of sufficient quantities of spermatocytes at specific stages of prophase I. Here, we describe a combination of methodologies we adapted for rapid and reliable isolation of synchronized fixed mouse spermatocytes. We show that chromatin isolated from these cells can be used to study chromatin-binding sites by ChIP-seq. High-quality data we obtained from INO80 ChIP-seq in zygotene cells was used for functional analysis of chromatin-binding sites.

Transcriptome data are insufficient to control false discoveries in regulatory network inference.

Inference of causal transcriptional regulatory networks (TRNs) from transcriptomic data suffers notoriously from false positives. Approaches to control the false discovery rate (FDR), for example, via permutation, bootstrapping, or multivariate Gaussian distributions, suffer from several complications: difficulty in distinguishing direct from indirect regulation, nonlinear effects, and causal structure inference requiring "causal sufficiency," meaning experiments that are free of any unmeasured, confounding variables. Here, we use a recently developed statistical framework, model-X knockoffs, to control the FDR while accounting for indirect effects, nonlinear dose-response, and user-provided covariates. We adjust the procedure to estimate the FDR correctly even when measured against incomplete gold standards. However, benchmarking against chromatin immunoprecipitation (ChIP) and other gold standards reveals higher observed than reported FDR. This indicates that unmeasured confounding is a major driver of FDR in TRN inference. A record of this paper's transparent peer review process is included in the supplemental information.

Glucocorticoid Receptor-Dependent Binding Analysis Using Chromatin Immunoprecipitation and Quantitative Polymerase Chain Reaction.

ChIP-qPCR offers the opportunity to identify interactions of DNA-binding proteins such as transcription factors and their respective DNA binding sites. Thereby, transcription factors can interfere with gene expression, resulting in up- or downregulation of their target genes. Utilizing ChIP, it is possible to identify specific DNA binding sites that are bound by the DNA-binding proteins in dependence on treatment or prevailing conditions. During ChIP, DNA-binding proteins are reversibly cross-linked to their DNA binding sites and the DNA itself is fragmented. Using bead-captured antibodies, the target proteins are isolated while still binding their respective DNA response element. Using quantitative PCR, these DNA fragments are amplified and quantified. In this protocol, DNA binding sites of the glucocorticoid receptor are identified by treatment with the synthetic glucocorticoid Dexamethasone in murine bone marrow-derived macrophages.

Chromatin Immunoprecipitation in Adipose Tissue and Adipocytes: How to Proceed and Optimize the Protocol for Transcription Factor DNA Binding.

Chromatin immunoprecipitation (ChIP) coupled to qPCR or sequencing is a crucial experiment to determine direct transcriptional regulation under the control of specific transcriptional factors or co-regulators at loci-specific or pan-genomic levels.Here we provide a reliable method for processing ChIP from adipocytes or frozen adipose tissue collection, isolation of nuclei, cross-linking of protein-DNA complexes, chromatin shearing, immunoprecipitation, and DNA purification. We also discuss critical steps for optimizing the experiment to perform a successful ChIP in lipid-rich cells/tissues.

Native ChIP: Studying the Genome-Wide Distribution of Histone Modifications in Cells and Tissue.

For the genome-wide mapping of histone modifications, chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing remains the benchmark method. While crosslinked ChIP can be used for all kinds of targets, native ChIP is predominantly used for strong and direct DNA interactors like histones and their modifications. Here we describe a native ChIP protocol that can be used for cells and tissue material.

Reverse Chromatin Immunoprecipitation (R-ChIP).

DNA-protein interactions play fundamental roles in diverse biological functions. The gene-centered method is used to identify the upstream regulators of defined genes. In this study, we developed a novel method for capturing the proteins that bind to certain chromatin fragments or DNA sequences, which is called reverse chromatin immunoprecipitation (R-ChIP). This technology uses a set of specific DNA probes labeled with biotin to isolate chromatin or DNA fragments, and the DNA-associated proteins are then analyzed using mass spectrometry. This method can capture DNA-associated proteins with sufficient quantity and purity for identification.

Bioinformatics Core Workflow for ChIP-Seq Data Analysis.

Chromatin immunoprecipitation (ChIP) followed by next-generation sequencing (-seq) has been the most common genomics method for studying DNA-protein interactions in the last decade. ChIP-seq technology became standard both experimentally and computationally. This chapter presents a core workflow that covers data processing and initial analytical steps of ChIP-seq data. We provide a step-by-step protocol of the commands as well as a fully assembled Snakemake workflow. Along the protocol, we discuss key tool parameters, quality control, output reports, and preliminary results.

Cleavage Under Targets and Release Using Nuclease (CUT&RUN) in Macrophages.

Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is a method to detect specific interactions between DNA and DNA-associated proteins. It is valuable for the characterization of the binding of transcription factors or co-regulators genome wide. Furthermore, it can be used for epigenetic profiling, chromatin accessibility assessment, and identification of regulatory elements. Compared to the more commonly used chromatin immunoprecipitation (ChIP), CUT&RUN has several advantages including an in situ approach as well as no need for sonication. However, the biggest advantage is the reduced cell amounts that are required for CUT&RUN, which makes it more attractive for experiments with limited cell numbers. In this chapter, we describe a reliable CUT&RUN protocol for macrophages that can be performed within 2 days and includes a library preparation so that the sample can be directly sequenced.

Differential Analysis of Protein-DNA Binding Using ChIP-Seq Data.

Chromatin immunoprecipitation in combination with next-generation sequencing (ChIP-Seq) allows probing of protein-DNA binding in a rapid and genome-wide fashion. Herein we describe the required steps to preprocess ChIP-Seq data and to analyze the differential binding of proteins to DNA for perturbation experiments. In these experiments, different conditions are compared to find the underlying biological mechanisms caused by the stimulus or treatment. In addition, we provide a sample analysis using the steps outlined in the chapter.

An Optimized High-Resolution Mapping Method for Glucocorticoid Receptor-DNA Binding in Mouse Primary Macrophages.

ChIP-exo is a powerful tool for achieving enhanced sensitivity and single-base-pair resolution of transcription factor (TF) binding, which utilizes a combination of chromatin immunoprecipitation (ChIP) and lambda exonuclease digestion (exo) followed by high-throughput sequencing. ChIP-nexus (chromatin immunoprecipitation experiments with nucleotide resolution through exonuclease, unique barcode, and single ligation) is an updated and simplified version of the original ChIP-exo method, which has reported an efficient adapter ligation through the DNA circularization step. Building upon an established method, we present a protocol for generating NGS (next-generation sequencing) ready and high-quality ChIP-nexus library for glucocorticoid receptor (GR). This method is specifically optimized for bone marrow-derived macrophage (BMDM) cells. The protocol is initiated by the formation of DNA-protein cross-links in intact cells. This is followed by chromatin shearing, chromatin immunoprecipitation, ligation of sequencing adapters, digestion of adapter-ligated DNA using lambda exonuclease, and purification of single-stranded DNA for circularization and library amplification.

Sequential ChIP-Seq.

ChIP-Seq has been used extensively to profile genome-wide transcription factor binding and post-translational histone modifications. A sequential ChIP assay determines the in vivo co-localization of two proteins to the same genomic locus. In this chapter, we combine the two protocols in Sequential ChIP-Seq, a method for identifying genome-wide sites of in vivo protein co-occupancy.

Estrogen Receptor Chromatin Profiling by CUT&RUN.

Gonadal steroid hormones, namely, testosterone, progesterone, and estrogens, influence the physiological state of an organism through the regulation of gene transcription. Steroid hormones activate nuclear hormone receptor (HR), transcription factors (TFs), which bind DNA in a tissue- and cell type-specific manner to influence cellular function. Identifying the genomic binding sites of HRs is essential to understanding mechanisms of hormone signaling across tissues and disease contexts. Traditionally, chromatin immunoprecipitation followed by sequencing (ChIP-seq) has been used to map the genomic binding of HRs in cancer cell lines and large tissues. However, ChIP-seq lacks the sensitivity to detect TF binding in small numbers of cells, such as genetically defined neuronal subtypes in the brain. Cleavage Under Targets & Release Under Nuclease (CUT&RUN) resolves most of the technical limitations of ChIP-seq, enabling the detection of protein-DNA interactions with as few as 100-1000 cells. In this chapter, we provide a stepwise CUT&RUN protocol for detecting and analyzing the genome-wide binding of estrogen receptor α (ERα) in mouse brain tissue. The steps described here can be used to identify the genomic binding sites of most TFs in the brain.

Cleavage Under Targets and Release Using Nuclease (CUT&RUN) of Epigenetic Regulators.

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) allows for the identification of genomic targeting of DNA-binding proteins. Cleavage Under Targets and Release Using Nuclease (CUT&RUN) modifies this process by including a nuclease to digest DNA around a protein of interest. The result is a higher signal-to-noise ratio and decreased required starting material. This allows for high-fidelity sequence identification from as few as 500 cells, enabling chromatin profiling of precious tissue samples or primary cell types, as well as less abundant chromatin-binding proteins: all at significantly increased throughput.

Integrative Analysis of CUT&Tag and RNA-Seq Data Through Bioinformatics: A Unified Workflow for Enhanced Insights.

Cleavage Under Targets and Tagmentation (CUT&Tag) is a recent methodology used for robust epigenomic profiling that, unlike conventional chromatin immunoprecipitation (ChIP-Seq), requires only a limited amount of cells as starting material. RNA sequencing (RNA-Seq) reveals the presence and quantity of RNA in a biological sample, describing the continuously changing cellular transcriptome. The integrated analysis of transcriptional activity, histone modifications, and chromatin accessibility via CUT&Tag is still in its infancy compared to the well-established ChIP-Seq. This chapter describes a robust bioinformatics methodology and workflow to perform an integrative CUT&Tag/RNA-Seq analysis.

Hepatitis B Virus Covalently Closed Circular DNA Chromatin Immunoprecipitation Assay.

Hepatitis B virus (HBV) is an obligate human hepatotropic DNA virus causing both transient and chronic infection. The livers of chronic hepatitis B patients have a high risk of developing liver fibrosis, cirrhosis, and hepatocellular carcinoma. The nuclear episomal viral DNA intermediate, covalently closed circular DNA (cccDNA), forms a highly stable complex with host and viral proteins to serve as a transcription template and support HBV infection chronicity. Thus, characterization of the composition and dynamics of cccDNA nucleoprotein complexes providing cccDNA stability and gene regulation is of high importance for both basic and medical research. The presented method for chromatin immunoprecipitation coupled with qPCR (ChIP-qPCR) allows to assess provisional physical interaction of the protein of interest (POI) with cccDNA using POI-specific antibody, the level of enrichment of a POI on cccDNA versus control/background is characterized quantitatively using qPCR.

ChIP-qPCR of FLAG-Tagged Proteins in Bacteria.

DNA-protein interactions occur in biological processes such as genome replication, gene transcription, DNA repair, and chromatin compaction and organization. Mapping the distribution of the DNA-bound proteins on the chromosome is essential for understanding their associated biological process. Chromatin immunoprecipitation (ChIP) involves the antibody-mediated enrichment of DNA fragments bound by a target protein and has become one of the most powerful techniques for exploring the distribution of proteins on the chromosome. By incorporating quantitative polymerase chain reaction (qPCR) downstream of the ChIP assay, ChIP-qPCR was developed to describe binding profiles of DNA-associated proteins at a candidate locus. In this chapter, we describe ChIP-qPCR. We provide a step-by-step protocol for the preparation of a ChIP library of a 3× FLAG-tagged protein in bacteria, describe how downstream qPCR experiments can be performed with the appropriate controls, and explain how the data is analyzed. This chapter provides reliable technical guidance for ChIP-qPCR studies in bacteria.

High-Resolution Characterization of DNA/Protein Complexes in Living Bacteria.

The occurrence of DNA looping is ubiquitous. This process plays a well-documented role in the regulation of prokaryotic gene expression, such as in regulation of the Escherichia coli lactose (lac) operon. Here we present two complementary methods for high-resolution in vivo detection of DNA/protein binding within the bacterial nucleoid by using either chromatin immunoprecipitation combined with phage λ exonuclease digestion (ChIP-exo) or chromatin endogenous cleavage (ChEC), coupled with ligation-mediated polymerase chain reaction (LM-PCR) and Southern blot analysis. As an example, we apply these in vivo protein-mapping methods to E. coli to show direct binding of architectural proteins in the Lac repressor-mediated DNA repression loop.

Chromatin Immunoprecipitation Using Imbibed Seeds of Arabidopsis thaliana.

Chromatin immunoprecipitation (ChIP) is used to analyze the targeting of a protein to a specific region of chromatin in vivo. Here, we present an instructive ChIP protocol for Arabidopsis imbibed seeds. The protocol covers all steps, from the sampling of imbibed seeds to the reverse crosslinking of immunoprecipitated protein-DNA complexes, and includes experimental tips and notes. The targeting of the protein to DNA is determined by quantitative PCR (qPCR) using reverse crosslinked DNA. The protocol can be further scaled up for ChIP-sequencing (ChIP-seq) analysis. As an example of the protocol, we include a ChIP-quantitative PCR (ChIP-qPCR) analysis demonstrating the targeting of PIF1 to the ABI5 promoter.

Summary of ChIP-Seq Methods and Description of an Optimized ChIP-Seq Protocol.

Chromatin immunoprecipitation (ChIP) is an invaluable method to characterize interactions between proteins and genomic DNA, such as the genomic localization of transcription factors and post-translational modification of histones. DNA and proteins are reversibly and covalently crosslinked using formaldehyde. Then the cells are lysed to release the chromatin. The chromatin is fragmented into smaller sizes either by micrococcal nuclease (MN) or sonication and then purified from other cellular components. The protein-DNA complexes are enriched by immunoprecipitation (IP) with antibodies that target the epitope of interest. The DNA is released from the proteins by heat and protease treatment, followed by degradation of contaminating RNAs with RNase. The resulting DNA is analyzed using various methods, including polymerase chain reaction (PCR), quantitative PCR (qPCR), or sequencing. This protocol outlines each of these steps for both yeast and human cells. This chapter includes a contextual discussion of the combination of ChIP with DNA analysis methods such as ChIP-on-Chip, ChIP-qPCR, and ChIP-Seq, recent updates on ChIP-Seq data analysis pipelines, complementary methods for identification of binding sites of DNA binding proteins, and additional protocol information about ChIP-qPCR and ChIP-Seq.