Springer protocols feed by Cell Biology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Deep Sequencing of Small Chromatin-Associated RNA: Bioinformatic Analysis
Chromatin-associated RNA (caRNA) is a newly identified class of RNA stably linked to chromatin and responsible for maintaining the higher order structure of euchromatic regions in an accessible form (Schubert et al., Mol Cell 48, 434 – 444, 2012). This section provides a pipeline of bioinformatic analysis for this specific type of RNA-Seq. It can be run locally by combining several scripts or be carried out on the Galaxy platform (Giardine et al., Genome Res 15:1451–1455, 2005). (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - October 30, 2013 Category: Cytology Source Type: news
Deep Sequencing of Small Chromatin-Associated RNA: Isolation and Library Preparation
Chromatin-associated RNA (caRNA) is a newly identified class of RNA molecules stably associated with chromatin, maintaining the higher order structure of euchromatic regions in an accessible form (Schubert et al., Mol Cell, doi:10.1016/j.molcel.2012.08.021, 2012). This section provides a detailed protocol describing the isolation of small caRNA from Drosophila cells and preparation of libraries suited for stranded small (20–200 bp) RNA deep sequencing on the Illumina platform. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - October 30, 2013 Category: Cytology Source Type: news
Purification of Specific Chromatin Domains from Single-Copy Gene Loci in Saccharomyces cerevisiae
Most methods currently available for the analysis of chromatin in vivo rely on a priori knowledge of putative chromatin components or their posttranslational modification state. The isolation of defined native chromosomal regions provides an attractive alternative to obtain a largely unbiased molecular description of chromatin. Here, we describe a strategy combining site-specific recombination at the chromosome with an efficient tandem affinity purification protocol to isolate a single-copy gene locus from the yeast Saccharomyces cerevisiae. The method allows robust enrichment of a targeted chromatin domain, making it amen...
Source: Springer protocols feed by Cell Biology - October 30, 2013 Category: Cytology Source Type: news
Analysis of Chromatin Composition of Repetitive Sequences: The ChIP-Chop Assay
Chromatin immunoprecipitation (ChIP) is a powerful method that allows to probe specific protein-DNA interactions in vivo and to estimate the occupancy of proteins at specific sites of the genome. However, the traditional ChIP assay is not able to distinguish whether repeats that share identical sequences display a different composition of associated factors and, consequently, different functions. The ChIP-chop method provides a useful application to analyze the interaction of proteins with repetitive sequences based on their CpG methylation content. The detailed ChIP-chop protocol that serves to determine the chromatin com...
Source: Springer protocols feed by Cell Biology - October 30, 2013 Category: Cytology Source Type: news
Chromatin Immunoprecipitation
Chromatin plays important functions in regulating many biological processes, including DNA transcription, replication, and repair. The use of chromatin immunoprecipitation (ChIP) assays has contributed enormously to identify interactions between DNA and a wide range of nuclear proteins including histones and their different posttranslational modifications as well as a variety of transcription factors. ChIP assays have been successfully used to map histone modifications and histone variants, as well as binding of transcription factors and chromatin-modifying complexes in both, specific candidate loci and the entire genome. ...
Source: Springer protocols feed by Cell Biology - October 30, 2013 Category: Cytology Source Type: news
Salt-Urea, Sulfopropyl-Sepharose, and Covalent Chromatography Methods for Histone Isolation and Fractionation
The histones are essential basic proteins intimately involved in most DNA-templated processes. Thus, their purification and fractionation for analysis and their use for in vitro chromatin transactions are of fundamental importance for understanding their role in chromatin structure and regulation of DNA functions. Here are described three new protocols for histone isolation from undisturbed whole cells. They avoid the conventional non-denaturing cell lysis, which affects the native posttranslational modifications of histones, and the cumbersome use of reverse-phase high-performance liquid chromatography. The three methodol...
Source: Springer protocols feed by Cell Biology - October 30, 2013 Category: Cytology Source Type: news
Analysis of Histone Posttranslational Modifications from Nucleolus-Associated Chromatin by Mass Spectrometry
Chromatin is unevenly distributed within the eukaryote nucleus and it contributes to the formation of morphologically and functionally distinct substructures, called chromatin domains and nuclear bodies. Here we describe an approach to assess specific chromatin features, the histone posttranslational modifications (PTMs), of the largest nuclear sub-compartment, the nucleolus. In this chapter, methods for the isolation of nucleolus-associated chromatin from native or formaldehyde-fixed cells and the effect of experimental procedures on the outcome of mass spectrometry analysis of histone PTMs are compared. (Source: Springer...
Source: Springer protocols feed by Cell Biology - October 30, 2013 Category: Cytology Source Type: news
Microscale Thermophoresis for the Assessment of Nuclear Protein-Binding Affinities
The rapid advance in our knowledge of cellular regulatory mechanisms, including those involving chromatin-based processes, stems in part from the development of biophysical techniques such as fluorescence spectroscopy, surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC). Despite their widespread utility, each of these techniques has its pros and cons, and new techniques are still required. Here we describe the application of microscale thermophoresis (MST), a novel technique based on thermophoresis, to characterize the binding between histone peptides and a histone chaperone protein, in free solutio...
Source: Springer protocols feed by Cell Biology - October 30, 2013 Category: Cytology Source Type: news
Hydroxymethylated DNA Immunoprecipitation (hmeDIP)
5-hydroxymethylcytosine (5hmC) was recently identified as an abundant epigenetic mark in mammals. Subsequent research has implicated 5hmC in normal mammalian development and disease pathogenesis in humans. Many of the techniques commonly used to assay for canonical 5-methylcytosine (5mC) cannot distinguish between 5hmC and 5mC. The development of antibodies specific to 5hmC has allowed for specific enrichment of DNA fragments containing 5hmC. Hydroxymethylated DNA immunoprecipitation (hmeDIP) has become an invaluable tool for determining both locus-specific and genome-wide profiles of 5hmC in mammalian DNA. Here, we descri...
Source: Springer protocols feed by Cell Biology - October 30, 2013 Category: Cytology Source Type: news
Predictive Binding Geometry of Ligands to DNA Minor Groove: Isohelicity and Hydrogen-Bonding Pattern
The interaction of drugs and dyes with nucleic acids, particularly when binding to DNA minor groove occurs, has increasing importance in biomedical sciences. This is due to the resulting biological activity and to the possibility of recognizing AT and GC base pairs. In such cases, DNA binding can be predicted if appropriate helical and hydrogen-bonding parameters are deduced from DNA models, and a simplified geometrical rule in the form of a stencil is then applied on computer-drawn molecules of interest. Relevant structure parameter values for minor groove binders are the length (4.6 < L < 5.4 Å) and angle (15...
Source: Springer protocols feed by Cell Biology - October 30, 2013 Category: Cytology Source Type: news
Investigating 5-Hydroxymethylcytosine (5hmC): The State of the Art
The discovery of 5-hydroxymethylcytosine (5hmC) as an abundant base in mammalian genomes has excited the field of epigenetics, and stimulated an intense period of research activity aimed at decoding its biological significance. However, initial research efforts were hampered by a lack of assays capable of specifically detecting 5hmC. Consequently, the last 3 years have seen the development of a plethora of new techniques designed to detect both global levels and locus-specific profiles of 5hmC in mammalian genomes. This research effort has culminated in the recent publication of two complementary techniques for quantitativ...
Source: Springer protocols feed by Cell Biology - October 30, 2013 Category: Cytology Source Type: news
Methyl-Combing: Single-Molecule Analysis of DNA Methylation on Stretched DNA Fibers
The methyl-combing technique combines the dynamic molecular combing method with the detection of DNA modifications. The assay allows the single-molecule analysis of epigenetic marks on regularly stretched DNA fibers, at the megabase scale with kilobase resolution. The protocol presented in this chapter is based on proof-of-principle experiments where the single-molecule detection of DNA methylation has been performed on unmethylated and in vitro methylated λ phage DNA. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - October 30, 2013 Category: Cytology Source Type: news
Standard DNA Methylation Analysis in Mouse Epidermis: Bisulfite Sequencing, Methylation-Specific PCR, and 5-Methyl-Cytosine (5mC) Immunological Detection
In mammals, methylation of cytosine C-5 position is a major heritable epigenetic mark on the DNA molecule. Maintenance of proper DNA methylation patterns is a key process during embryo development and in the maintenance of adult tissue homeostasis. The use of experimental procedures based on the chemical modification of cytosine by sodium bisulfite and the development of antibodies recognizing 5mC have essentially contributed to our knowledge on DNA methylation dynamics in normal and disease states. Here we describe standard procedures for bisulfite sequencing, methylation-specific PCR, and 5mC immunodetection using mouse ...
Source: Springer protocols feed by Cell Biology - October 30, 2013 Category: Cytology Source Type: news
Combined Bidimensional Electrophoresis and Electron Microscopy to Study Specific Plasmid DNA Replication Intermediates in Human Cells
We describe how to use this experimental system to run preparative agarose 2D-gel and to extract specific replication intermediates to visualize by electron microscopy. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - October 30, 2013 Category: Cytology Source Type: news
Visualization and Interpretation of Eukaryotic DNA Replication Intermediates In Vivo by Electron Microscopy
The detailed understanding of the DNA replication process requires structural insight. The combination of psoralen cross-linking and electron microscopy has been extensively exploited to reveal the fine architecture of in vivo DNA replication intermediates. This approach proved instrumental to uncover the basic mechanisms of DNA duplication, as well as the perturbation of this process by various forms of replication stress. The replication structures are stabilized in vivo (by psoralen cross-linking) prior to extraction and enrichment procedures, allowing their visualization at the transmission electron microscope. This ch...
Source: Springer protocols feed by Cell Biology - October 30, 2013 Category: Cytology Source Type: news
