Willadsengrimes8683

A hallmark feature of active cis-regulatory elements (CREs) in eukaryotes is their nucleosomal depletion and, accordingly, higher accessibility to enzymatic treatment. This property has been the basis of a number of sequencing-based assays for genome-wide identification and tracking the activity of CREs across different biological conditions, such as DNAse-seq, ATAC-seq , NOMeseq, and others. However, the fragmentation of DNA inherent to many of these assays and the limited read length of short-read sequencing platforms have so far not allowed the simultaneous measurement of the chromatin accessibility state of CREs located distally from each other. The combination of labeling accessible DNA with DNA modifications and nanopore sequencing has made it possible to develop such assays. Here, we provide a detailed protocol for carrying out the SMAC-seq assay (Single-Molecule long-read Accessible Chromatin mapping sequencing), in its m6A-SMAC-seq and m6A-CpG-GpC-SMAC-seq variants, together with methods for data processing and analysis, and discuss key experimental and analytical considerations for working with SMAC-seq datasets.The ATAC-seq method enables the genome-wide analysis of accessible chromatin revealing transcriptionally active and poised regulatory elements. The ATAC-seq analysis of clinical specimens at a single-cell resolution reveals the cellular composition of the tissue contributing to the understanding of intra-tissue heterogeneity. Here we describe our method for nuclei isolation from frozen specimens with wide applicability across tissue types, producing nuclei suitable for a number of molecular profiling methods including ATAC-seq in bulk and at a single-cell resolution.Bulk chromatin encompasses complex sets of histone posttranslational modifications (PTMs) that recruit (or repel) the diverse reader domains of Chromatin-Associated Proteins (CAPs) to regulate genome processes (e.g., gene expression, DNA repair, mitotic transmission). The binding preference of reader domains for their PTMs mediates localization and functional output, and are often dysregulated in disease. As such, understanding chromatin interactions may lead to novel therapeutic strategies, However the immense chemical diversity of histone PTMs, combined with low-throughput, variable, and nonquantitative methods, has defied accurate CAP characterization. This chapter provides a detailed protocol for dCypher, a novel approach for the rapid, quantitative interrogation of CAPs (as mono- or multivalent Queries) against large panels (10s to 100s) of PTM-defined histone peptide and semisynthetic nucleosomes (the potential Targets). We describe key optimization steps and controls to generate robust binding data. Further, we compare the utility of histone peptide and nucleosome substrates in CAP studies, outlining important considerations in experimental design and data interpretation.Several methods have been developed to map protein-DNA interactions genome-wide in the last decades. Protein A-DamID (pA-DamID) is a recent addition to this list with distinct advantages. pA-DamID relies on antibody-based targeting of the bacterial Dam enzyme, resulting in adenine methylation of DNA in contact with the protein of interest. This m6A can then be visualized by microscopy, or mapped genome-wide. The main advantages of pA-DamID are an easy and direct visualization of DNA that is in contact with the protein of interest, unbiased mapping of protein-DNA interactions, and the possibility to select specific subpopulations of cells by flow cytometry before further sample processing. pA-DamID is particularly suited to study proteins that form large chromatin domains or that are part of distinct nuclear structures such as the nuclear lamina. This chapter describes the pA-DamID procedure from cell harvesting to the preparation of microscopy slides and high-throughput sequencing libraries.Targeted DamID (TaDa) is a means of profiling the binding of any DNA-associated protein cell-type specifically, including transcription factors, RNA polymerase, and chromatin-modifying proteins. The technique is highly sensitive, highly reproducible, requires no mechanical disruption, cell isolation or antibody purification, and can be performed by anyone with basic molecular biology knowledge. Here, we describe the TaDa method and downstream bioinformatics data processing.In mammalian cells, multiprotein complexes form at specific genomic regulatory elements (REs) to control gene expression, which in turn is ultimately responsible for cellular identity. Consequently, insight into the molecular composition of these regulatory complexes is of major importance for our understanding of any physiological or pathological cellular state or transition. However, it remains extremely difficult to identify the protein complex(es) assembled at a specific RE in the mammalian genome using conventional approaches. We therefore developed a novel single locus isolation technique based on Transcription Activator-Like Effector (TALE) proteins termed TALE-mediated isolation of nuclear chromatin (TINC). When coupled with high-resolution mass spectrometry, TINC enables the identification and characterization of protein complexes formed at any RE of interest. Using the Nanog promoter in mouse embryonic stem cells as proof of concept, this chapter describes in detail the novel TINC methodology as well as subsequent mass spectrometric considerations.Single-particle tracking (SPT) makes it possible to directly observe single protein diffusion dynamics in living cells over time. Thus, SPT has emerged as a powerful method to quantify the dynamics of nuclear proteins such as transcription factors (TFs). Here, we provide a protocol for conducting and analyzing SPT experiments with a focus on fast tracking ("fastSPT") of TFs in mammalian cells. First, we explore how to engineer and prepare cells for SPT experiments. Next, we examine how to optimize SPT experiments by imaging at low densities to minimize tracking errors and by using stroboscopic excitation to minimize motion-blur. Next, we discuss how to convert raw SPT data into single-particle trajectories. Finally, we illustrate how to analyze these trajectories using the kinetic modeling package Spot-On. We discuss how to use Spot-On to fit histograms of displacements and extract useful information such as the fraction of TFs that are bound and freely diffusing, and their associated diffusion coefficients.The genome in a eukaryotic cell is packaged into chromatin and regulated by chromatin-binding and chromatin-modifying factors. Many of these factors and their complexes have been identified before, but how each genomic locus interacts with its surrounding proteins in the nucleus over time and in changing conditions remains poorly described. Measuring protein-DNA interactions at a specific locus in the genome is challenging and current techniques such as capture of a locus followed by mass spectrometry require high levels of enrichment. Epi-Decoder, a method developed in budding yeast, enables systematic decoding of the proteome of a single genomic locus of interest without the need for locus enrichment. Instead, Epi-Decoder uses massive parallel chromatin immunoprecipitation of tagged proteins combined with barcoding a genomic locus and counting of coimmunoprecipitated barcodes by DNA sequencing (TAG-ChIP-Barcode-Seq). In this scenario, DNA barcode counts serve as a quantitative readout for protein binding of each tagged protein to the barcoded locus. Epi-Decoder can be applied to determine the protein-DNA interactions at a wide range of genomic loci, such as coding genes, noncoding genes, and intergenic regions. Furthermore, Epi-Decoder provides the option to study protein-DNA interactions upon changing cellular and/or genetic conditions. In this protocol, we describe in detail how to construct Epi-Decoder libraries and how to perform an Epi-Decoder analysis.The investigation of cohesin binding sites throughout different mammalian genomes by ChIP-sequencing has been fundamental to discover how cohesin and CTCF collaborate to form chromatin loops and to gain insight in the intricate regulation of cohesin. Here we describe a detailed ChIP protocol that has been successfully used for different cohesin subunits and cohesin regulators in various cell lines.Chromatin immunoprecipitation and sequencing (ChIP-seq) is a well-established method to study the epigenetic profile at the genome-wide scale, including histone modifications and DNA-protein interactions. click here It provides valuable insights to better understand disease mechanisms. Here we present an optimized ChIP-seq protocol suitable for human cardiac tissues, especially the frozen biobanked small biopsy samples.Whole-genome bisulfite sequencing (WGBS) is currently the gold standard for DNA methylation (5-methylcytosine, 5mC) profiling; however, the destructive nature of sodium bisulfite results in DNA fragmentation and subsequent biases in sequencing data. Such issues have led to the development of bisulfite-free methods for 5mC detection. Nanopore sequencing is a long read nondestructive approach that directly analyzes DNA and RNA fragments in real time. Recently, computational tools have been developed that enable base-resolution detection of 5mC from Oxford Nanopore sequencing data. In this chapter, we provide a detailed protocol for preparation, sequencing, read assembly, and analysis of genome-wide 5mC using Nanopore sequencing technologies.DNA methylation is an epigenetic modification with an established role in both normal cellular function and mammalian disease. Despite well-characterized associations between aberrant DNA methylation changes and gene expression, evidence for a causal relationship in this context has been difficult to obtain. Early techniques for interrogating the role of DNA methylation in the regulation of gene transcription lack specificity and, where more specific techniques such and ZNFs and TALEs have been developed, they are limited by their extensive cost and labor requirements. However, the recent advent of CRISPR-based technologies has revolutionized our potential for site-specific epigenomic editing. Here, we provide a detailed protocol for the design, construction, and utilization of a transient, CRISPR-based DNA methylation-editing system in mammalian cells.Bisulfite sequencing is the "gold-standard" technique for DNA methylation analysis. By combining bisulfite sequencing with high-throughput, next-generation sequencing technology, we can document methylation from many thousands of individual reads (equivalent to alleles or "cells"), for multiple target regions and from many samples simultaneously. Here, we describe a next-generation bisulfite-sequencing assay for targeted DNA methylation analysis which offers scope for the simultaneous interrogation of multiple genomic loci across numerous samples.Array-based EWAS have become an increasingly popular technique to identify population epigenetic effects, particularly in humans. With the arrival of nonhuman species arrays, such as the mouse, this is likely to become an even more widely used technology. This chapter provides the less experienced researcher a guide to the analysis of data from the most widely used platform, the Illumina Infinium Methylation assay. This includes an overview of quality filtering, data normalization, analysis options, and techniques to improve the interpretation of results.