PAN-INTACT enables isolation of lineage-specific nuclei from fibrous tissues
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ABSTRACT:
PROVIDER: PRJNA490328 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7818890_1.fastq.gz | Fastqsanger.gz | |||
| SRR7818890_2.fastq.gz | Fastqsanger.gz | |||
| SRR7818891_1.fastq.gz | Fastqsanger.gz | |||
| SRR7818891_2.fastq.gz | Fastqsanger.gz | |||
| SRR7818892_1.fastq.gz | Fastqsanger.gz |
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Project description:PAN-INTACT method can be used to isolate cell type specific nuclei from fibrous mouse organs, which are particularly problematic. It is broadly applicable for profiling the transcriptional and epigenetic landscape of specific cell types. We envision that our method can be used to systematically probe mechanistic details of cell type-specific functions within individual organs of intact mice.
2019-04-05 | GSE119792 | GEO
Project description:Recent studies have highlighted the extraordinary cell type diversity that exists within mammalian organs, yet the molecular drivers of such heterogeneity remain elusive. To address this issue, much attention has been focused on profiling the transcriptome and epigenome of individual cell types. However, standard cell type isolation methods based on surface or fluorescent markers remain problematic for cells residing within organs with significant connective tissue. Since the nucleus contains both genomic and transcriptomic information, the isolation of nuclei tagged in specific cell types (INTACT) method provides an attractive solution. Although INTACT has been successfully applied to plants, flies, zebrafish, frogs, and mouse brain and adipose tissue, broad use across mammalian organs remains challenging. Here we describe the PAN-INTACT method, which can be used to isolate cell type specific nuclei from fibrous mouse organs, which are particularly problematic. As a proof-of-concept, we demonstrate successful isolation of cell type-specific nuclei from the mouse heart, which contains substantial connective tissue and harbors multiple cell types, including cardiomyocytes, fibroblasts, endothelial cells, and epicardial cells. Compared to established techniques, PAN-INTACT allows more rapid isolation of cardiac nuclei to facilitate downstream applications. We show cell type-specific isolation of nuclei from the hearts of Nkx2-5Cre/+; R26Sun1-2xsf-GFP-6xmyc/+ mice, which we confirm by expression of lineage markers. Furthermore, we perform Assay for Transposase Accessible Chromatin (ATAC)-Seq to provide high-fidelity chromatin accessibility maps of Nkx2-5+ nuclei. To extend the applicability of PAN-INTACT, we also demonstrate successful isolation of Wt1+ podocytes from adult kidney. Taken together, our data suggest that PAN-INTACT is broadly applicable for profiling the transcriptional and epigenetic landscape of specific cell types. Thus, we envision that our method can be used to systematically probe mechanistic details of cell type-specific functions within individual organs of intact mice.
| S-EPMC6445515 | biostudies-literature
Project description:In this study we used transgenic mouse model to comapre two isolation techniques INTACT and FANS for the isolation of activated neuronal nuclei. Comparison is perfomed on multiple levels like isolation efficiency, membrane integrity, transcriptional and epigenetic state using RNA-seq and ATAC-seq
2021-01-28 | GSE165644 | GEO
Project description:Ultra-parallel ChIP-seq by barcoding of intact nuclei
| PRJNA435556 | ENA
Project description:INTACT vs. FANS for cell-type-specific nuclei isolation: A comprehensive qualitative and quantitative comparison
| PRJNA695261 | ENA
Project description:We introduce a more sensitive method for purifying specific cell-types from small numbers of fly heads for epigenomic analysis: a method we call ‘mini-INTACT’. We modified the INTACT method in Drosophila [Henry G. L. et al., Nucleic Acids Research, 2012, 1–14] to enable purification of genetically tagged rare cell types from Drosophila brain requiring ~50-100 fold less material. We used male fruit flies as a model to examine the effects of social-isolation vs. social-enrichment on the epigenome and transcriptome of dopaminergic neurons defined by expression of the TH-GAL4 driver. Using ChIP-seq on mini-INTACT purified nuclei, RNA-seq and bioinformatic analysis, we identify regulation of the transcriptome and behavior by Activity Related Genes who’s expression is induced by social enrichment.
2019-02-07 | GSE126186 | GEO
Project description:Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) is an invaluable tool for mapping chromatin-associated proteins. Processing of samples still remains largely individual and labor-intensive, hindering the assay throughput and comparability across samples. Here we present a novel method for ultra-parallelized high-throughput ChIP-seq for the systematic mapping of histone modifications and transcription factors. The method, called RELACS (Restriction Enzyme-based Labeling of Chromatin in Situ), barcodes chromatin within intact nuclei extracted from different tissutal sources. Barcoded nuclei are pooled and processed within the same ChIP, for maximal comparability and drastical workload reduction. The choice of user-friendly, straightforward, enzymatic steps for chromatin fragmentation and barcoding makes RELACS particularly suitable for implementation in any clinical laboratory settings, for scarce samples, and large-scale studies.
2018-03-06 | GSE111000 | GEO
Project description:Due to their abundance in the oceans, their extraordinary biodiversity and the increasing use for biotech applications, the study of diatom biology is receiving more and more attention in the recent years. One of the limitations in developing molecular tools for diatoms lies in the peculiar nature of their cell wall, that is made of silica and organic molecules and that hinders the application of standard methods for cell lysis required, for example, to extract organelles. In this study we present a protocol for intact nuclei isolation from diatoms that was successfully applied to three different species: two pennates, Pseudo-nitzschia multistriata and Phaeodactylum tricornutum, and one centric diatom species, Chaetoceros diadema. Intact nuclei were extracted by treatment with acidified NH4F solution combined to low intensity sonication pulses and separated from cell debris via FAC-sorting upon incubation with SYBR Green. Microscopy observations confirmed the integrity of isolated nuclei and high sensitivity DNA electrophoresis showed that genomic DNA extracted from isolated nuclei has low degree of fragmentation. This protocol has proved to be a flexible and versatile method to obtain intact nuclei preparations from different diatom species and it has the potential to speed up applications such as epigenetic explorations as well as single cell ("single nuclei") genomics, transcriptomics and proteomics in different diatom species.
| S-EPMC7813820 | biostudies-literature
Project description:Cells organize their actions partly through tightly controlled protein-protein interactions – collectively termed the interactome. Here we use crosslinking mass spectrometry (XL-MS) to chart the interactome of intact human nuclei. We overall identified ~8700 crosslinks, of which 2/3 represent links connecting distinct proteins. From this data we constructed an overview of the nuclear interactome. We observed that the histone proteins on the nucleosomes expose well-defined crosslinking hot-spots. For several nucleosome-interacting proteins, such as USF3 and Ran GTPase, the data allowed us to build models of their binding mode to the nucleosome. For HMGN2 the data guided the construction of a refined model of the interaction with the nucleosome, based on complementary NMR, XL-MS and modeling. Excitingly, several isoform-specific interactors seem to exist for distinct histone H1 variants and the analysis of crosslinks carrying post-translational modifications allowed us to extract how specific modifications influence the nucleosome interactome. Overall, our data depository will support future structural and functional analysis of cell nuclei, including the nucleoprotein assemblies they harbor.
2018-07-23 | PXD007513 | Pride
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