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.

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.

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.

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.

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.

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.

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.

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.

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.

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.