INTACT method for gene expression and chromatin profiling of individual cell types within a tissue
Ontology highlight
ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
PROVIDER: GSE19654 | GEO | 2010/06/14
SECONDARY ACCESSION(S): PRJNA122553
REPOSITORIES: GEO
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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
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
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 All experiments were done using strepavidin pulldown DNA cohybridized with total input DNA to the same array. Two channels per array, Cy5 and Cy3, were used in each experiment.
2010-05-27 | E-GEOD-19788 | biostudies-arrayexpress
Project description:We developed scNanoSeq-CUT&Tag, a streamlined method by adapting a modified CUT&Tag protocol to Oxford Nanopore sequencing platform for efficient chromatin modification profiling at single-cell resolution. We firstly tested the performance of scNanoSeq-CUT&Tag on six human cell lines: K562, 293T, GM12878, HG002, H9, HFF1 and adult mouse blood cells, it showed that scNanoSeq-CUT&Tag can accurately distinguish different cell types in vitro and in vivo. Moreover, scNanoSeq-CUT&Tag enables to effectively map the allele-specific epigenomic modifications in the human genome andallows to analyze co-occupancy of histone modifications. Taking advantage of long-read sequencing,scNanoSeq-CUT&Tag can sensitively detect epigenomic state of repetitive elements. In addition, by applying scNanoSeq-CUT&Tag to testicular cells of adult mouse B6D2F1, we demonstrated that scNanoSeq-CUT&Tag maps dynamic epigenetic state changes during mouse spermatogenesis. Finally, we exploited the epigenetic changes of human leukemia cell line K562 during DNA demethylation, it showed that NanoSeq-CUT&Tag can capture H3K27ac signals changes along DNA demethylation. Overall, we prove that scNanoSeq-CUT&Tag is a valuable tool for efficiently probing chromatin state changes within individual cells.
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Project description:Any average pattern observed at the population level (cross-sectional analysis) may confound two different types of processes: some processes that occur among individuals and others that occur within individuals. Separating within- from among-individual processes is critical for our understanding of ecological and evolutionary dynamics. The within-individual centring method allows distinguishing within- from among-individual processes and this method has been widely used in ecology to investigate both linear and quadratic patterns. Here we show that two alternative equations could be used for the investigation of quadratic within-individual patterns. We explain the different assumptions and constraints of both equations. Reviewing the literature, we found that mainly one of these two equations has been used in studies investigating quadratic patterns. Yet this equation might not be the most appropriate in all circumstances leading to bias and imprecision. We show that these two alternative equations make different assumptions about the shape of the within-individual pattern. One equation assumes that the within-individual effect is related to an absolute process whereas the other assumes the effect arises from an individual relative process. The choice of using one equation instead of the other should depend upon the biological process investigated. Using simulations, we showed that a mismatch between the assumptions made by the equation used to analyse the data and the biological process investigated might led to flawed inference affecting output of model selection and accuracy of estimates. We stress that the equation used should be chosen carefully. We provide step by step guidelines for choosing an equation when studying quadratic pattern with the within-individual centring approach. We encourage the use of the within-individual centring method, promoting its relevant application for nonlinear relationships.
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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
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
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
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
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