The Experts below are selected from a list of 4938 Experts worldwide ranked by ideXlab platform
Job Dekker - One of the best experts on this subject based on the ideXlab platform.
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Randomized ligation control for Chromosome Conformation Capture.
Cold Spring Harbor protocols, 2015Co-Authors: Jon-matthew Belton, Job DekkerAbstract:In experiments using Chromosome Conformation Capture followed by PCR (3C-PCR) or Chromosome Conformation Capture carbon copy (5C), it is critical to control for intrinsic biases in the restriction fragments of interest and the probes or primers used for detection. Characteristics such as GC%, annealing temperature, efficiency of 3C primers or 5C probes, and length of restriction fragment can cause variations in primer or probe performance and fragment ligation efficiency. Bias can be measured empirically by production of a random control library, as described here, to be used with the 3C library of interest.
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Chromosome Conformation Capture Carbon Copy (5C) in Budding Yeast
Cold Spring Harbor protocols, 2015Co-Authors: Jon-matthew Belton, Job DekkerAbstract:Chromosome Conformation Capture carbon copy (5C) is a high-throughput method for detecting ligation products of interest in a Chromosome Conformation Capture (3C) library. 5C uses ligation-mediated amplification (LMA) to generate carbon copies of 3C ligation product junctions using single-stranded oligonucleotide probes. This procedure produces a 5C library of short DNA molecules which represent the interactions between the corresponding restriction fragments. The 5C library can be amplified using universal primers containing the Illumina paired-end adaptor sequences for subsequent high-throughput sequencing.
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Chromosome Conformation Capture (3C) in Budding Yeast.
Cold Spring Harbor protocols, 2015Co-Authors: Jon-matthew Belton, Job DekkerAbstract:Chromosome Conformation Capture (3C) is a method for studying chromosomal organization that takes advantage of formaldehyde cross-linking to measure the spatial association of two pieces of chromatin. The 3C method begins with whole-cell formaldehyde fixation of chromatin. After cell lysis, solubilized chromatin is digested with a type II restriction endonuclease, and cross-linked DNA fragmentsareligatedtogether.Cross-linksarereversed bydegradationwithproteinaseK,andchimeric DNA molecules are purified by standard phenol:chloroform extraction. The resulting 3C library represents chromatin fragments that may be separated by large genomic distances or located on different Chromosomes, but are close enough in three-dimensional space for cross-linking. Locus-specific oligonucleotide primers are used to detect interactions of interest in the 3C library using end-point polymerase chain reaction (PCR).
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Two ways to fold the genome during the cell cycle: insights obtained with Chromosome Conformation Capture
Epigenetics & Chromatin, 2014Co-Authors: Job DekkerAbstract:Genetic and epigenetic inheritance through mitosis is critical for dividing cells to maintain their state. This process occurs in the context of large-scale re-organization of Chromosome Conformation during prophase leading to the formation of mitotic Chromosomes, and during the reformation of the interphase nucleus during telophase and early G1. This review highlights how recent studies over the last 5 years employing Chromosome Conformation Capture combined with classical models of Chromosome organization based on decades of microscopic observations, are providing new insights into the three-dimensional organization of chromatin inside the interphase nucleus and within mitotic Chromosomes. One striking observation is that interphase genome organization displays cell type-specific features that are related to cell type-specific gene expression, whereas mitotic Chromosome folding appears universal and tissue invariant. This raises the question of whether or not there is a need for an epigenetic memory for genome folding. Herein, the two different folding states of mammalian genomes are reviewed and then models are discussed wherein instructions for cell type-specific genome folding are locally encoded in the linear genome and transmitted through mitosis, e.g., as open chromatin sites with or without continuous binding of transcription factors. In the next cell cycle these instructions are used to re-assemble protein complexes on regulatory elements which then drive three-dimensional folding of the genome from the bottom up through local action and self-assembly into higher order levels of cell type-specific organization. In this model, no explicit epigenetic memory for cell type-specific Chromosome folding is required.
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Analysis of long-range chromatin interactions using Chromosome Conformation Capture
Methods (San Diego Calif.), 2012Co-Authors: Natalia Naumova, Ye Zhan, Emily Smith, Job DekkerAbstract:Chromosome Conformation Capture, or 3C, is a pioneering method for investigating the three-dimensional structure of chromatin. 3C is used to analyze long-range looping interactions between any pair of selected genomic loci. Most 3C studies focus on defined genomic regions of interest that can be up to several hundred Kb in size. The method has become widely adopted and has been modified to increase throughput to allow unbiased genome-wide analysis. These large-scale adaptations are presented in other articles in this issue of Methods. Here we describe the 3C procedure in detail, including the appropriate use of the technology, the experimental set-up, an optimized protocol and troubleshooting guide, and considerations for data analysis. The protocol described here contains previously unpublished improvements, which save time and reduce labor. We pay special attention to primer design, appropriate controls and data analysis. We include notes and discussion based on our extensive experience to help researchers understand the principles of 3C-based techniques and to avoid common pitfalls and mistakes. This paper represents a complete resource and detailed guide for anyone who desires to perform 3C.
Stefan Grob - One of the best experts on this subject based on the ideXlab platform.
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improved brassica rapa reference genome by single molecule sequencing and Chromosome Conformation Capture technologies
Horticulture research, 2018Co-Authors: Lei Zhang, Stefan Grob, Xuming Li, Jianli Liang, Song Li, Jian Wu, Feng Cheng, Lin Cheng, Fan Wang, Wencai YangAbstract:Brassica rapa comprises several important cultivated vegetables and oil crops. Current reference genome assemblies of Brassica rapa are quite fragmented and not highly contiguous, thereby limiting extensive genetic and genomic analyses. Here, we report an improved assembly of the B. rapa genome (v3.0) using single-molecule sequencing, optical mapping, and Chromosome Conformation Capture technologies (Hi-C). Relative to the previous reference genomes, our assembly features a contig N50 size of 1.45 Mb, representing a ~30-fold improvement. We also identified a new event that occurred in the B. rapa genome ~1.2 million years ago, when a long terminal repeat retrotransposon (LTR-RT) expanded. Further analysis refined the relationship of genome blocks and accurately located the centromeres in the B. rapa genome. The B. rapa genome v3.0 will serve as an important community resource for future genetic and genomic studies in B. rapa. This resource will facilitate breeding efforts in B. rapa, as well as comparative genomic analysis with other Brassica species.
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Technical Review: A Hitchhiker's Guide to Chromosome Conformation Capture.
Methods in Molecular Biology, 2018Co-Authors: Stefan Grob, Giacomo CavalliAbstract:The introduction of Chromosome Conformation Capture (3C) technologies boosted the field of 3D-genome research and significantly enhanced the available toolset to study chromosomal architecture. 3C technologies not only offer increased resolution compared to the previously dominant cytological approaches but also allow the simultaneous study of genome-wide 3D chromatin contacts, thereby enabling a candidate-free perspective on 3D-genome architecture. Since its introduction in 2002, 3C technologies evolved rapidly and now constitute a collection of tools, each with their strengths and pitfalls with respect to specific research questions. This chapter aims at guiding 3C novices through the labyrinth of potential applications of the various family members, hopefully providing a valuable basis for choosing the appropriate strategy for different research questions.
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Technical Review: A Hitchhiker’s Guide to Chromosome Conformation Capture
Methods in molecular biology (Clifton N.J.), 2017Co-Authors: Stefan Grob, Giacomo CavalliAbstract:The introduction of Chromosome Conformation Capture (3C) technologies boosted the field of 3D-genome research and significantly enhanced the available toolset to study chromosomal architecture. 3C technologies not only offer increased resolution compared to the previously dominant cytological approaches but also allow the simultaneous study of genome-wide 3D chromatin contacts, thereby enabling a candidate-free perspective on 3D-genome architecture. Since its introduction in 2002, 3C technologies evolved rapidly and now constitute a collection of tools, each with their strengths and pitfalls with respect to specific research questions. This chapter aims at guiding 3C novices through the labyrinth of potential applications of the various family members, hopefully providing a valuable basis for choosing the appropriate strategy for different research questions.
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Circular Chromosome Conformation Capture in Plants.
Methods in molecular biology (Clifton N.J.), 2017Co-Authors: Stefan GrobAbstract:The study of nuclear architecture promises novel insights into genome function and regulation. Hereby, quantitative methods based on Chromosome Conformation Capture (3C) revolutionized the field, as they allow accurate and unbiased characterization of 3D genome organization of genomic regions of interest. The choice of the appropriate 3C derivate is crucial to acquire results suited for a specific research question. Circular 3C (4C) is the method of choice to study the genome-wide 3D architecture of a specific genomic region of interest. Here, I present a robust 4C protocol, established in Arabidopsis thaliana, which can be employed by any experienced molecular biologist and is applicable in various other plant species.
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Chromosome Conformation Capture-based studies reveal novel features of plant nuclear architecture.
Current opinion in plant biology, 2017Co-Authors: Stefan Grob, Ueli GrossniklausAbstract:Nuclear genome organization has recently received increasing attention due to its manifold functions in basic nuclear processes, such as replication, transcription, and the maintenance of genome integrity. Using technologies based on Chromosome Conformation Capture, such as Hi-C, we now have the possibility to study the three-dimensional organization of the genome at unprecedented resolution, shedding light onto a previously unexplored level of nuclear architecture. In plants, research in this field is still in its infancy but a number of publications provided first insights into basic principles of nuclear genome organization and the factors that influence it. Apart from general aspects, newly discovered three-dimensional Conformations, such as the KNOT, raise special interest on how nuclear organization may influence the function of the genome in previously unexpected ways.
Tom Sexton - One of the best experts on this subject based on the ideXlab platform.
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4C-Seq: Interrogating Chromatin Looping with Circular Chromosome Conformation Capture.
Methods in molecular biology (Clifton N.J.), 2020Co-Authors: Nezih Karasu, Tom SextonAbstract: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.
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Detecting Spatial Chromatin Organization by Chromosome Conformation Capture II: Genome-Wide Profiling by Hi-C.
Methods in molecular biology (Clifton N.J.), 2016Co-Authors: Matteo Vietri Rudan, Suzana Hadjur, Tom SextonAbstract:The Chromosome Conformation Capture (3C) method has been invaluable in studying chromatin interactions in a population of cells at a resolution surpassing that of light microscopy, for example in the detection of functional contacts between enhancers and promoters. Recent developments in sequencing-based chromosomal contact mapping (Hi-C, 5C and 4C-Seq) have allowed researchers to interrogate pairwise chromatin interactions on a wider scale, shedding light on the three-dimensional organization of Chromosomes. These methods present significant technical and bioinformatic challenges to consider at the start of the project. Here, we describe two alternative methods for Hi-C, depending on the size of the genome, and discuss the major computational approaches to convert the raw sequencing data into meaningful models of how genomes are organized.
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Sensitive detection of chromatin coassociations using enhanced Chromosome Conformation Capture on chip
Nature Protocols, 2012Co-Authors: Tom Sexton, Sreenivasulu Kurukuti, Jennifer A Mitchell, David Umlauf, Takashi Nagano, Peter FraserAbstract:Chromosome Conformation Capture (3C) is a powerful technique for analyzing spatial chromatin organization in vivo. Technical variants of the assay ('4C') allow the systematic detection of genome-wide coassociations with bait sequences of interest, enabling the nuclear environments of specific genes to be probed. We describe enhanced 4C (e4C, enhanced Chromosome Conformation Capture on chip), a technique incorporating additional enrichment steps for bait-specific sequences, and thus improving sensitivity in the detection of weaker, distal chromatin coassociations. In brief, e4C entails the fixation, restriction digestion and ligation steps of conventional 3C, with an optional chromatin immunoprecipitation (ChIP) step to select for subsets of chromatin coassociations, followed by bait enrichment by biotinylated primer extension and pull-down, adapter ligation and PCR amplification. Chromatin coassociations with the bait sequence can then be assessed by hybridizing e4C products to microarrays or sequencing. The e4C procedure takes approximately 1 week to go from tissue to DNA ready for microarray hybridization.
Luc Gaudreau - One of the best experts on this subject based on the ideXlab platform.
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Detection of Short-Range DNA Interactions in Mammalian Cells Using High-Resolution Circular Chromosome Conformation Capture Coupled to Deep Sequencing.
Methods in molecular biology (Clifton N.J.), 2015Co-Authors: Jean-françois Millau, Luc GaudreauAbstract:DNA interactions shape the genome to physically and functionally connect regulatory elements to their target genes. Studying these interactions is crucial to understanding the molecular mechanisms that regulate gene expression. In this chapter, we present a protocol for high-resolution circular Chromosome Conformation Capture coupled to deep sequencing. This methodology allows to investigate short-range DNA interactions (
Ralph Stadhouders - One of the best experts on this subject based on the ideXlab platform.
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Unbiased Interrogation of 3D Genome Topology Using Chromosome Conformation Capture Coupled to High-Throughput Sequencing (4C-Seq).
Methods in molecular biology (Clifton N.J.), 2016Co-Authors: Rutger W W Brouwer, Mirjam C. G. N. Van Den Hout, Wilfred F. J. Van Ijcken, Eric Soler, Ralph StadhoudersAbstract:The development and widespread implementation of Chromosome Conformation Capture (3C) technology has allowed unprecedented new insight into how Chromosomes are folded in three-dimensional (3D) space. 3C and its derivatives have contributed tremendously to the now widely accepted view that genome topology plays an important role in many major cellular processes, at a Chromosome-wide scale, but certainly also at the level of individual genetic loci. A particularly popular application of 3C technology is to study transcriptional regulation, allowing researchers to draw maps of gene regulatory connections beyond the linear genome through addition of the third dimension. In this chapter, we provide a highly detailed protocol describing 3C coupled to high-throughput sequencing (referred to as 3C-Seq or more commonly 4C-Seq), allowing the unbiased interrogation of genome-wide chromatin interactions with specific genomic regions of interest. Interactions between spatially clustered DNA fragments are revealed by crosslinking the cells with formaldehyde, digesting the genome with a restriction endonuclease and performing a proximity ligation step to link interacting genomic fragments. Next, interactions with a selected DNA fragment are extracted from the 3C library through a second round of digestion and ligation followed by an inverse PCR. The generated products are immediately compatible with high-throughput sequencing, and amplicons from different PCR reactions can easily be multiplexed to dramatically increase throughput. Finally, we provide suggestions for data analysis and visualization.
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r3Cseq: an R/Bioconductor package for the discovery of long-range genomic interactions from Chromosome Conformation Capture and next-generation sequencing data
Nucleic acids research, 2013Co-Authors: Supat Thongjuea, Ralph Stadhouders, Frank Grosveld, Eric Soler, Boris LenhardAbstract:The coupling of Chromosome Conformation Capture (3C) with next-generation sequencing technologies enables the high-throughput detection of long-range genomic interactions, via the generation of ligation products between DNA sequences, which are closely juxtaposed in vivo. These interactions involve promoter regions, enhancers and other regulatory and structural elements of Chromosomes and can reveal key details of the regulation of gene expression. 3C-seq is a variant of the method for the detection of interactions between one chosen genomic element (viewpoint) and the rest of the genome. We present r3Cseq, an R/Bioconductor package designed to perform 3C-seq data analysis in a number of different experimental designs. The package reads a common aligned read input format, provides data normalization, allows the visualization of candidate interaction regions and detects statistically significant chromatin interactions, thus greatly facilitating hypothesis generation and the interpretation of experimental results. We further demonstrate its use on a series of real-world applications.
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Multiplexed Chromosome Conformation Capture sequencing for rapid genome-scale high-resolution detection of long-range chromatin interactions
Nature Protocols, 2013Co-Authors: Ralph Stadhouders, Petros Kolovos, Rutger Brouwer, Jessica Zuin, Anita Van Den Heuvel, Christel Kockx, Robert-jan Palstra, Kerstin S Wendt, Frank Grosveld, Wilfred Van IjckenAbstract:Chromosome Conformation Capture (3C) technology is a powerful and increasingly popular tool for analyzing the spatial organization of genomes. Several 3C variants have been developed (e.g., 4C, 5C, ChIA-PET, Hi-C), allowing large-scale mapping of long-range genomic interactions. Here we describe multiplexed 3C sequencing (3C-seq), a 4C variant coupled to next-generation sequencing, allowing genome-scale detection of long-range interactions with candidate regions. Compared with several other available techniques, 3C-seq offers a superior resolution (typically single restriction fragment resolution; approximately 1–8 kb on average) and can be applied in a semi-high-throughput fashion. It allows the assessment of long-range interactions of up to 192 genes or regions of interest in parallel by multiplexing library sequencing. This renders multiplexed 3C-seq an inexpensive, quick (total hands-on time of 2 weeks) and efficient method that is ideal for the in-depth analysis of complex genetic loci. The preparation of multiplexed 3C-seq libraries can be performed by any investigator with basic skills in molecular biology techniques. Data analysis requires basic expertise in bioinformatics and in Linux and Python environments. The protocol describes all materials, critical steps and bioinformatics tools required for successful application of 3C-seq technology.