Springer protocols feed by Genetics/Genomics 
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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.The Cruciform DNA Mobility Shift Assay: A Tool to Study Proteins That Recognize Bent DNA
So-called architectural DNA-binding proteins such as those of the HMGB-box family induce DNA bending and kinking. However, these proteins often display only a weak sequence preference, making the analysis of their DNA-binding characteristics difficult if not impossible in a standard electrophoretic mobility shift assay (EMSA). In contrast, such proteins often bind prebent DNAs with high affinity and specificity. A synthetic cruciform DNA structure will often provide an ideal binding site for such proteins, allowing their affinities for both bent and linear DNAs to be directly and simply determined by a modified form of EMS...
Source: Springer protocols feed by Genetics/Genomics - September 15, 2015 Category: Genetics & Stem Cells Source Type: news
Precise Identification of DNA-Binding Proteins Genomic Location by Exonuclease Coupled Chromatin Immunoprecipitation (ChIP-exo)
DNA-binding proteins play a crucial role in all living organisms by interacting with various DNA sequences across the genome. While several methods have been used to study the interaction between DNA and proteins in vitro, chromatin immunoprecipitation followed by sequencing (ChIP-seq) has become the standard technique for identifying the genome-wide location of DNA-binding proteins in vivo. However, the resolution of standard ChIP-seq methodology is limited by the DNA fragmentation process and presence of contaminating DNA. A significant improvement of the ChIP-seq technique results from the addition of an exonuclease tre...
Source: Springer protocols feed by Genetics/Genomics - September 15, 2015 Category: Genetics & Stem Cells Source Type: news
Analysis of DNA Supercoiling Induced by DNA–Protein Interactions
Certain DNA-interacting proteins induce a pronounced bending in the double helix and cause topological stresses that are compensated by the formation of supercoils in DNA. Such supercoils, when forming on a circular plasmid, give rise to a series of topoisomers that run at different speeds during electrophoresis. The number of supercoils introduced in the plasmid can provide information on the protein; it can, for example, help determine the number of nucleosomes that are assembled on the plasmid or indicate whether the DNA-bending activity of a transcription factor is important enough to cause a topological stress. Becaus...
Source: Springer protocols feed by Genetics/Genomics - September 15, 2015 Category: Genetics & Stem Cells Source Type: news
siRNA Screening of Nuclear Proteins
Functional gene screening is a basic strategy for identifying candidate genes that are responsible for biological processes of interest to researchers. RNA-mediated interference (RNAi)-based screening is one such approach. Although the knockdown technique using siRNA is widely accepted, careful evaluation is required to avoid false positive candidates as much as possible, because various cellular responses can be caused by events other than the target gene disruption. In this chapter, we introduce a method for a relatively small-scale siRNA screening that is capable of finding RNA binding proteins that regulate a specific ...
Source: Springer protocols feed by Genetics/Genomics - January 1, 2015 Category: Genetics & Stem Cells Source Type: news
Knockdown of Nuclear-Retained Long Noncoding RNAs Using Modified DNA Antisense Oligonucleotides
Long noncoding RNAs (lncRNAs) have recently emerged as important players in diverse cellular processes. Among them, a large fraction of lncRNAs are localized within cell nucleus. And several of these nuclear-retained lncRNAs have been found to regulate key nuclear processes, which brings up the requirement of effective genetic tools to explore the functions of this “dark matter” inside the nucleus. While siRNAs and shRNAs are widely used tools in loss-of-function studies, their general efficiency in depleting nuclear-retained lncRNAs is limited, due to the fact that the RNAi machinery is located mainly in the c...
Source: Springer protocols feed by Genetics/Genomics - January 1, 2015 Category: Genetics & Stem Cells Source Type: news
Genome-Wide Analysis of Long Noncoding RNA Turnover
Genome-wide analysis for determining RNA turnover is an advanced method in RNA biology that examines the specific half-life of nuclear noncoding RNA (ncRNA). In particular, a pulse-labeling method using uridine analogs enables the determination of RNA stability under physiologically undisturbed conditions. The technique involves pulse labeling of endogenous RNAs in mammalian cells with 5′-bromo-uridine (BrU), followed by measuring the chronological decrease of BrU-labeled RNAs using deep sequencing. The method is called BrU immunoprecipitation chase assay (BRIC) or BRIC through deep sequencing (BRIC-seq). Here, we de...
Source: Springer protocols feed by Genetics/Genomics - January 1, 2015 Category: Genetics & Stem Cells Source Type: news
Reconstitution of Nucleocytoplasmic Transport Using Digitonin-Permeabilized Cells
Nucleocytoplasmic transport is crucial not only for basic cellular activities but also for the physiological adaptation of cells to various environmental stimuli that affect development, cell-fate determination, or disease development. The basic transport mechanisms have been revealed during the past two decades through the identification and biochemical characterizations of factors mediating the transport, dissecting the transport process and examining the function of nuclear pore complexes (NPCs). In this chapter, we describe methods for a nuclear transport reconstitution assay using digitonin-permeabilized mammalian cel...
Source: Springer protocols feed by Genetics/Genomics - January 1, 2015 Category: Genetics & Stem Cells Source Type: news
MMCT-Mediated Chromosome Engineering Technique Applicable to Functional Analysis of lncRNA and Nuclear Dynamics
Recent evidence implicated several long noncoding RNA (lncRNA) in gene expression in cis or trans through regulating the local chromosomal architecture. However, the mechanisms underlying the lncRNA mediated silencing of multiple genes remain unknown. We believe that Microcell Mediated Chromosome Transfer (MMCT) is a suitable approach for functional analysis of lncRNAs and nuclear dynamics. MMCT is a unique research technique that can be generally used to transfer a single chromosome from one mammalian cell to another. Transferred chromosomes can be stably maintained as functioning in the recipient cells. Since there is no...
Source: Springer protocols feed by Genetics/Genomics - January 1, 2015 Category: Genetics & Stem Cells Source Type: news
Purification of Noncoding RNA and Bound Proteins Using FLAG Peptide-Conjugated Antisense-Oligonucleotides
To understand the function of certain RNAs, including noncoding RNAs, it is important to identify the proteins that interact with the RNAs. Here we describe the method for purification of ribonucleoprotein (RNP) complexes composed of specific cellular RNAs by pull-down with FLAG peptide-conjugated antisense oligonucleotide (ASO). Using this method, we identified a novel protein component of U7 snRNP complex. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - January 1, 2015 Category: Genetics & Stem Cells Source Type: news
Cross-Linking and Immunoprecipitation of Nuclear RNA-Binding Proteins
The systematic identification of in vivo targets of nuclear RNA-binding proteins (RBPs) is crucial to elucidate the physiological functions of each RBP. However, it has been difficult to distinguish real targets from nonspecifically bound RNAs and to determine the exact binding sites of each RBP by using a conventional RNA-immunoprecipitation (RIP) method. Photoactivatable Ribonucleoside-Enhanced Cross-linking and Immunoprecipitation (PAR-CLIP) is a recently developed method that relies on RNA-protein cross-linking to reduce the contamination of nonspecifically bound RNAs. Furthermore, in combination with high-throughput s...
Source: Springer protocols feed by Genetics/Genomics - January 1, 2015 Category: Genetics & Stem Cells Source Type: news
Studying RNA-Binding Protein Interactions with Target mRNAs in Eukaryotic Cells: Native Ribonucleoprotein Immunoprecipitation (RIP) Assays
Post-transcriptional regulation of mRNA can potently dictate protein expression patterns in eukaryotic cells. This mode of regulation occurs through cis-acting regulatory regions in the mRNA transcript that mediate direct interactions with trans-acting RNA-binding proteins (RBPs). This mRNA/protein interaction can be studied in numerous ways that range from in vitro to in vivo through messenger ribonucleoprotein immunoprecipitation (mRNP-IP or RIP) assays. This modified immunoprecipitation approach is an important and sensitive method to determine the regulation of gene expression by specific RBPs under different cellular ...
Source: Springer protocols feed by Genetics/Genomics - January 1, 2015 Category: Genetics & Stem Cells Source Type: news
Extracting, Enriching, and Identifying Nuclear Body Sub-Complexes Using Label-Based Quantitative Mass Spectrometry
Determining the proteome of a nuclear body is a crucial step toward understanding its function; however, it is extremely challenging to obtain pure nuclear body preparations. Moreover, many nuclear proteins dynamically associate with multiple bodies and subnuclear compartments, confounding analysis. We have found that a more practical approach is to carry out affinity purification of nuclear body sub-complexes via the use of tagged nuclear-body-specific marker proteins. Here we describe in detail the method to identify new nuclear body protein sub-complexes through SILAC (stable isotope labeling by amino acids in culture)-...
Source: Springer protocols feed by Genetics/Genomics - January 1, 2015 Category: Genetics & Stem Cells Source Type: news
In Situ Dissection of RNA Functional Subunits by Domain-Specific Chromatin Isolation by RNA Purification (dChIRP)
Here we describe domain-specific chromatin isolation by RNA purification (dChIRP), a technique for dissecting the functional domains of a target RNA in situ. For an RNA of interest, dChIRP can identify domain-level intramolecular and intermolecular RNA–RNA, RNA–protein, and RNA–DNA interactions and maps the RNA’s genomic binding sites with higher precision than domain-agnostic methods. We illustrate how this technique has been applied to the roX1 lncRNA to resolve its domain-level architecture, discover its protein- and chromatin-interacting domains, and map its occupancy on the X chromosome. (Sourc...
Source: Springer protocols feed by Genetics/Genomics - January 1, 2015 Category: Genetics & Stem Cells Source Type: news
RNA Antisense Purification (RAP) for Mapping RNA Interactions with Chromatin
RNA-centric biochemical purification is a general approach for studying the functions and mechanisms of noncoding RNAs. Here, we describe the experimental procedures for RNA antisense purification (RAP), a method for selective purification of endogenous RNA complexes from cell extracts that enables mapping of RNA interactions with chromatin. In RAP, the user cross-links cells to fix endogenous RNA complexes and purifies these complexes through hybrid capture with biotinylated antisense oligos. DNA loci that interact with the target RNA are identified using high-throughput DNA sequencing. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - January 1, 2015 Category: Genetics & Stem Cells Source Type: news
Purification of Specific Chromatin Regions Using Oligonucleotides: Capture Hybridization Analysis of RNA Targets (CHART)
Identification of genomic binding sites and proteins associated with noncoding RNAs will lead to more complete mechanistic characterization of the regulatory activities of noncoding RNAs. Capture hybridization analysis of RNA targets (CHART) is a powerful technique wherein specific RNA molecules are isolated from cross-linked nuclear extracts using complementary, biotinylated capture oligonucleotides, allowing subsequent identification of genomic DNA and proteins cross-linked to the RNA of interest. Here, we describe the procedure for CHART and list strategies to optimize nuclear extract preparation, capture oligonucleotid...
Source: Springer protocols feed by Genetics/Genomics - January 1, 2015 Category: Genetics & Stem Cells Source Type: news
