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INTACT method for gene expression and chromatin profiling of individual cell types within a tissue

ABSTRACT: We used in vivo biotin labeling of a nuclear envelope protein in individual cell types followed by affinity isolation of labeled nuclei to measure gene expression and chromatin features of the hair and non-hair cell types of the Arabidopsis root epidermis. Keywords: Chromatin affinity-purification on microarray

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE19654 | GEO | 2010/06/14

SECONDARY ACCESSION(S): PRJNA122553

REPOSITORIES: GEO

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INTACT method for gene expression and chromatin profiling of individual cell types within a tissue

Project description:We used in vivo biotin labeling of a nuclear envelope protein in individual cell types followed by affinity isolation of labeled nuclei to measure gene expression and chromatin features of the hair and non-hair cell types of the Arabidopsis root epidermis. Keywords: Chromatin affinity-purification on microarray All experiments were done using two channels per chip. Two types of experiments were performed. Expression profiling was performed by comparing cDNAs synthesized from nuclear RNA isolated from INTACT-prepared Arabidopsis nuclei to total sonicated genomic DNA isolated from Arabidopsis plants. Chromatin profiling was performed using INTACT-prepared Arabidopsis nuclei. DNA isolated by Chromatin immunoprecipitation (ChIP) using antibodies to modified histones was compared to histone H3 ChIP DNA from the same sample.

2010-06-13 | E-GEOD-19654 | biostudies-arrayexpress

CATCH-IT method for measuring nucleosome turnover

Project description:Direct measurement of nucleosome turnover dynamics by using co-translational incorporation of the methionine (Met) surrogate azidohomoalaine (Aha) into proteins and subsequent ligation of biotin to Aha-containing proteins through the [3+2] cycloaddition reaction between the azide group of Aha and an alkyne linked to biotin. To measure turnover rates, we treat cells briefly with Aha, couple biotin to nucleosomes containing newly incorporated histones, affinity purify with strepavidin, wash stringently to remove non-histone proteins and H2A/H2B dimers, and analyze the affinity-purified DNA using tiling microarrays. We call this strategy 'CATCH-IT' for Covalent Attachment of Tags to Capture Histones and Identify Turnover. Keywords: Chromatin affinity-purification on microarray

2010-05-27 | GSE19788 | GEO

CATCH-IT method for measuring nucleosome turnover

2010-05-27 | E-GEOD-19788 | biostudies-arrayexpress

scNanoHi-C: a single-cell long-read concatemer sequencing method to reveal high-order structures within individual cells

Project description:High-order three-dimensional (3D) organization of regulatory elements provide a topological basis for genes regulation, but it remains unclear that how multiple regulatory elements interact across mammalian genome in a single cell. To address this, herein, we developed scNanoHi-C, which applies Nanopore sequencing to explore genome-wide proximal high-order chromatin contacts within individual cells. Evaluation of the method suggested that scNanoHi-C reliably and effectively profiled chromosome structure and distinguished structure subtype among single cells, which could also be used to detect genomic variations including CNVs and SVs, as well as scaffold the de novo assembly of single cell genomes. Importantly, our results suggested that high-order structures were extensively existed in active transcription chromatin across the genome, and multiway interactions between enhancers and their target promoters were observed in the first time within single cells. Taken together, scNanoHi-C sparked new insights of high-order 3D genome structure at the single-cell level.

2023-07-14 | GSE217189 | GEO

Distinguishing within- from between-individual effects: How to use the within-individual centring method for quadratic patterns.

Project description: Not available

| S-EPMC9298145 | biostudies-literature

Genome-wide localization of the RNA polymerase III transcription machinery in human cells

Project description:To characterize the pol III transcriptome in actively growing human cells, we used MR90hTeIrt lung fibroblasts, which are immortal but not transformed, with an intact Rb pathway. After formaldehyde cross-linking, chromatin was extracted and submitted to immunoprecipitation. To identify actively transcribed RNAP-III transcription units, we used antibodies directed against the RPC4 subunit of RNAP-III. To identify promoter regions, we used antibodies directed against Bdp1, which is part of both Brf1-TFIIIB and Brf2-TFIIIB, and thus should mark all types of active RNAP-III promoters. Antibodies directed against Brf1 were used to mark type 1 and 2 promoters. For type 3 promoters, we engineered an IMR90hTert cell line expressing the SNAPc subunit SNAP45 tagged at its C-terminal end with the biotin acceptor domain as well as the biotin ligase BirA and performed “chromatin affinity purification” (ChAP) with streptavidin beads. In each case, the precipitated material was then processed for deep sequencing.

2010-02-11 | GSE18184 | GEO

A native chromatin purification system for epigenomic profiling in C. elegans

Project description:We describe an in vivo chromatin purification system for genome-wide epigenetic profiling in C. elegans. In this system, we coexpressed the E. coli biotin ligase enzyme (BirA), together with the C. elegans H3.3 gene fused to BioTag, a 23-amino acid peptide serving as a biotinylation substrate for BirA, in vivo in worms. We developed methods to isolate chromatin under different salt extraction conditions, followed by affinity purification of biotinylated chromatin with streptavidin and genome-wide profiling with microarrays. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf All experiments were done using two channels per chip, comparing DNAs extracted from either salt-extracted or insoluble chromatin to whole nuclear chromatin, whole nuclear chromatin to randomly fragmented genomic DNA, streptavidin-bound biotin-tagged histone-variant-containing chromatin to salt-extracted chromatin, gel-purified mononucleosomes to whole EDTA-extracted soluble chromatin, or streptavidin-bound biotin-tagged histone-variant-containing chromatin to whole EDTA-extracted soluble chromatin to randomly fragmented DNA from embryo nuceli.

2010-05-19 | E-GEOD-18898 | biostudies-arrayexpress

Tissue-specific nuclei purification from animal models for genome-wide expression and chromatin profiling.

Project description:Here we describe a method for purifying nuclei from specific tissues of animal models that allows simultaneous analysis of both expression and chromatin profiles. The method is based on in vivo labeling of the nuclear envelope and subsequent affinity purification. We describe the use of the method to isolate nuclei from muscle. We determined genes preferentially expressed in muscle cells of adult C. elegans by analyzing RNA from total and affinity-puridied muscle nuclei using microarrays. We also determined the nucleosome occupancy in these nuclei by micrococcal nuclease mapping and paired-end sequencing.

2011-12-05 | E-GEOD-31602 | biostudies-arrayexpress

Biotinylation by antibody recognition - A novel method for proximity labeling

Project description:Identification of protein-protein interactions is a major goal of biological research. Despite technical advances over the last two decades, important but still largely unsolved challenges include the high-throughput detection of interactions directly from primary tissue and the identification of interactors of insoluble proteins that form higher-order structures. We have developed a novel, proximity-based labeling approach that uses antibodies to guide biotin deposition onto adjacent proteins in fixed cells and primary tissues. We showed our method to be specific and sensitive by labeling a mitochondrial matrix protein. Next, we used this method to profile the dynamic interactome of lamin A/C in multiple cell and tissue types under various treatment conditions. Our results suggest a considerable variation in the composition of the nuclear envelope of different tissues. Of note, DNA damage response proteins Ku70 and Ku80 are more abundant in the vicinity of lamin A/C after thermal stress. The ability to detect proximal proteins and putative interactors in intact tissues, and to compare affinities quantitatively under different conditions or in the presence of disease mutations, can provide a new window into cell biology and disease pathogenesis.

2017-10-17 | PXD004736 | Pride

Tissue-specific nuclei purification from animal models for genome-wide expression and chromatin profiling.

2011-12-05 | GSE31602 | GEO

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