Chromatin immunoprecipitation (ChIP) is used to probe the presence of proteins and/or their posttranslational modifications on genomic DNA. G140 supplier This method is often used alongside chromosome conformation capture approaches to obtain a better-rounded view of the functional relationship between chromatin architecture and its landscape. Since the inception of ChIP, its protocol has been modified to improve speed, sensitivity, and specificity. Combining ChIP with deep sequencing has recently improved its throughput and made genome-wide profiling possible. However, genome-wide analysis is not always the best option, particularly when many samples are required to study a given genomic region or when quantitative data is desired. We recently developed carbon copy-ChIP (2C-ChIP), a new form of the high-throughput ChIP analysis method ideally suited for these types of studies. 2C-ChIP applies ligation-mediated amplification (LMA) followed by deep sequencing to quantitatively detect specified genomic regions in ChIP samples. Here, we describe the generation of 2C-ChIP libraries and computational processing of the resulting sequencing data.The chromatin organization in the 3D nuclear space is essential for genome functionality. This spatial organization encompasses different topologies at diverse scale lengths with chromosomes occupying distinct volumes and individual chromosomes folding into compartments, inside which the chromatin fiber is packed in large domains (as the topologically associating domains, TADs) and forms short-range interactions (as enhancer-promoter loops). The widespread adoption of high-throughput techniques derived from chromosome conformation capture (3C) has been instrumental in investigating the nuclear organization of chromatin. In particular, Hi-C has the potential to achieve the most comprehensive characterization of chromatin 3D structures, as in principle it can detect any pair of restriction fragments connected as a result of ligation by proximity. However, the analysis of the enormous amount of genomic data produced by Hi-C techniques requires the application of complex, multistep computational procedures that may constitute a difficult task also for expert computational biologists. In this chapter, we describe the computational analysis of Hi-C data obtained from the lymphoblastoid cell line GM12878, detailing the processing of raw data, the generation and normalization of the Hi-C contact map, the detection of TADs and chromatin interactions, and their visualization and annotation.Within the nucleus, precise DNA folding and organization is mandatory for a tight control of gene expression. In the past 20 years, a wealth of molecular approaches has unraveled the existence of DNA territories. With the emergence of affordable deep-sequencing approaches, “Cs” techniques such as 4C, 5C, and HiC, to name a few, are now routinely performed by the scientific community in a large number of model systems. We have modified the HiC approach to a capture probe-based version named C-HiC. This updated assay has resulted in an improved throughput analysis, reduced input material, and good repeatability. The protocol described below details our procedure and notes for a C-HiC approach, designed to target only specific portion of a given genome.Technology advance during the past decade has greatly expanded our understanding of the higher-order structure of the genome. The various chromosome conformation capture (3C)-based techniques such as Hi-C have provided the most widely used tools for interrogating three-dimensional (3D) genome organization. We recently developed a Hi-C variant, DNase Hi-C, for characterizing 3D genome organization. DNase Hi-C employs DNase I for chromatin fragmentation, aiming to overcome restriction enzyme digestion-related limitations associated with traditional Hi-C methods. By combining DNase Hi-C with DNA capture technology, we further implemented a high-throughput approach, called targeted DNase Hi-C, which enables to map fine-scale chromatin architecture at exceptionally high resolution and thereby is an ideal tool for mapping the physical landscapes of cis-regulatory networks and for characterizing phenotype-associated chromatin 3D signatures. Here, I describe a detailed protocol of targeted DNase Hi-C library preparation, which covers experimental steps starting from cell cross-linking to library amplification.Chromatin Conformation Capture techniques have unveiled several layers of chromosome organization such as the segregation in compartments, the folding in topologically associating domains (TADs), and site-specific looping interactions. The discovery of this genome hierarchical organization emerged from the computational analysis of chromatin capture data. With the increasing availability of such data, automatic pipelines for the robust comparison, grouping, and classification of multiple experiments are needed. Here we present a pipeline based on the TADbit framework that emphasizes reproducibility, automation, quality check, and statistical robustness. This comprehensive modular pipeline covers all the steps from the sequencing products to the visualization of reconstructed 3D models of the chromatin.Chromosome conformation capture and its variants have allowed chromatin topology to be interrogated at a superior resolution and throughput than by microscopic methods. Among the method derivatives, 4C-seq (circular chromosome conformation capture, coupled to high-throughput sequencing) is a versatile, cost-effective means of assessing all chromatin interactions with a specific genomic region of interest, making it particularly suitable for interrogating chromatin looping events. We present the principles and procedures for designing and implementing successful 4C-seq experiments.The discovery of the DNA double helix by Watson and Crick in 1953 was the first report showing that the genomic information is not contained in a stretched linear molecule. After that, a huge advance in the knowledge of the structure of the eukaryotic genome in the nuclear space has been made over the last decades, bringing us to the widely accepted concept that the genome is packaged into hierarchical levels of higher-order three-dimensional structures. The spatial organization of the eukaryotic genome has direct influence on fundamental nuclear processes that include transcription, replication, and DNA repair. The idea that structural alterations of chromosomes may cause disease goes back to the early nineteenth century. Big effort has been devoted to the study of the three-dimensional architecture of the genome and its functional implications. In this chapter, I will describe the chromosome conformation capture (3C), one of the first techniques used to detect and measure the frequency of interactions between genomic sequences that are kept in spatial proximity in the nucleus.