TU-tagging: cell type specific RNA isolation from intact complex tissues
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ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE15445 | GEO | 2009/04/24
SECONDARY ACCESSION(S): PRJNA115809
REPOSITORIES: GEO
Similar Datasets
Project description:We show that the combination of spatially restricted uracil phosphoribosyltransferase (UPRT) expression with 4-thiouracil (4TU) delivery can be used to label and purify cell type specific RNA from intact complex tissues. This method is useful for isolating RNA from cell types that are difficult to isolate by dissection or dissociation methods and should work in any organism where UPRT can be spatially expressed, including mammals and other vertebrates. To confirm that thio-labeled RNA was from UPRT-expressing cells and demonstrate the utility of TU-tagging for cell type-specific RNA isolation, we purified TU-tagged and untagged RNA and compared them using microarrays. We chose to isolate RNA from larval glia, which are present in low number, are highly dispersed, and have a complex cell morphology, making them one of the most difficult cell types to isolate by dissection or dissociation methods. We used reversed polarity (repo)-GAL4 to drive expression of UAS-UPRT specifically in glial cells of the larval brain. We purified TU-tagged and untagged RNA from 72-96h larval brains, and hybridized them to custom Agilent microarrays. We performed two biological replicate experiments.
2010-06-19 | E-GEOD-15445 | biostudies-arrayexpress
Project description:To understand 4-TU labelling kinetics in a novel zebrafish transgenic line (lf:UPRT), we exposed adult lf:UPRT zebrafish (whereby animals have hepatocyte-specific uracil phosphoribosyltransferase expression) with 4-TU for 1, 3, 6 and 9 hours. We then dissected adult livers for SLAM-ITseq to determine the optimal 4-TU labelling time in our SLAM-ITseq workflow to allow us to study hepatocyte-specific nascent transcriptional changes.
2024-01-13 | GSE230830 | GEO
Project description:This SuperSeries is composed of the following subset Series: GSE2946: Synthesis vs. Abundance, Bradyzoite Development GSE2947: Thiouracil Pulse-chase, Tachyzoites and Bradyzoites mRNA stability data GSE2948: UPRT Transgenic cells, Human Arrays Abstract: Standard microarrays measure mRNA abundance, not mRNA synthesis, and therefore cannot identify the mechanisms that regulate gene expression. We have developed a method to overcome this limitation by using the salvage enzyme uracil phosphoribosyltransferase (UPRT) from the protozoan Toxoplasma gondii. T. gondii UPRT has been well characterized because of its application in monitoring parasite growth: mammals lack this enzyme activity and thus only the parasite incorporates (3)H-uracil into its nucleic acids. In this study we used RNA labeling by UPRT to determine the roles of mRNA synthesis and decay in the control of gene expression during T. gondii asexual development. We also used this approach to specifically label parasite RNA during a mouse infection and to incorporate thio-substituted uridines into the RNA of human cells engineered to express T. gondii UPRT, indicating that engineered UPRT expression will allow cell-specific analysis of gene expression in organisms other than T. gondii. Refer to individual Series
2006-03-07 | E-GEOD-4401 | biostudies-arrayexpress
Project description:adt12-01_uprt - plant rabt n°1 - Impact of RNA labeling by Plant RABT on plant transcriptome - Determine the incidence of RNA marking by the RABT method Plantation on the plants transcriptome. In several studies Clearly et al. have described a method referred as "4TU tagging" which can be use to study mRNA synthesis and decay either in a mixed population of cells or in a specific cell type (Cleary, Meiering et al. 2005, Zeiner, Cleary et al. 2008, Miller, Robinson et al. 2009, Rabani et al., 2011). Through the specific cell type expression in drosophila and mammalian cells of theToxoplasma gondii uracil PhosphoribosylTransferase activity and the metabolization of the uracil analog 4-thiouracil (4 TU), mRNA were selectively tagged, purified and used in microarray based analysis. 6 dye-swap - treated vs untreated comparison
2014-09-10 | E-GEOD-60532 | biostudies-arrayexpress
Project description:We developed a knock-in mouse line that combines four enrichment tools to study gene regulatory networks of the brain and other complex tissues. As a result, chromatin, nascent RNA, translating mRNA and mature miRNA can be purified from specific cell populations. Specifically, we developed "Tagger" – a Cre and/or Flp recombinase-dependent mouse model that expresses four proteins to enable purification of distinct populations of cellular nucleic acids from specific cell types. Upon activation by cell type-directed recombinases, cells of a desired population express: 1) exogenous uracil phoshoribosyltransferase (UPRT) for tagging nascent total RNA with thiol groups upon administration of 4-thiouracil (4TU, TU-Tag), 2) epitope-tagged large ribosomal subunit protein 22 (Rpl22) to co-immunoprecipitate (co-IP) ribosome-bound mRNA (RiboTag), 3) epitope-tagged Argonaute 2 (Ago2, a component of the RNA-induced silencing complex) to co-IP mature miRNA (AgoTag), and 4) nucleus-directed mKate2 red fluorescent protein, providing good tissue penetrating transmission properties for in vivo imaging and a fluorescent nuclear tag (NucTag) for FACS enrichment of nuclei for chromatin studies.
2019-10-30 | GSE123421 | GEO
Project description:adt12-01_uprt - plant rabt n°1 - Impact of RNA labeling by Plant RABT on plant transcriptome - Determine the incidence of RNA marking by the RABT method Plantation on the plants transcriptome. In several studies Clearly et al. have described a method referred as "4TU tagging" which can be use to study mRNA synthesis and decay either in a mixed population of cells or in a specific cell type (Cleary, Meiering et al. 2005, Zeiner, Cleary et al. 2008, Miller, Robinson et al. 2009, Rabani et al., 2011). Through the specific cell type expression in drosophila and mammalian cells of theToxoplasma gondii uracil PhosphoribosylTransferase activity and the metabolization of the uracil analog 4-thiouracil (4 TU), mRNA were selectively tagged, purified and used in microarray based analysis.
2014-09-10 | GSE60532 | GEO
Project description:Total RNA was extracted from about 10, 1-week or 5-weeks old, entire or dissected (head, legs, wings and ovaries removed) adult flies, using TRIzol reagent (Invitrogen) following manufacturer’s instructions. The following genotypes dedicated for TU-tagging cell - specific transcript isolation were used: Hand>LacZ;HA-UPRT, Hand>MblRNAi;HA-UPRT, Hand>Bru3;HA-UPRT and Hand>960CTG;HA-UPRT
2018-07-18 | GSE109370 | GEO
Project description:Abstract: Standard microarrays measure mRNA abundance, not mRNA synthesis, and therefore cannot identify the mechanisms that regulate gene expression. We have developed a method to overcome this limitation by using the salvage enzyme uracil phosphoribosyltransferase (UPRT) from the protozoan Toxoplasma gondii. T. gondii UPRT has been well characterized because of its application in monitoring parasite growth: mammals lack this enzyme activity and thus only the parasite incorporates (3)H-uracil into its nucleic acids. In this study we used RNA labeling by UPRT to determine the roles of mRNA synthesis and decay in the control of gene expression during T. gondii asexual development. We also used this approach to specifically label parasite RNA during a mouse infection and to incorporate thio-substituted uridines into the RNA of human cells engineered to express T. gondii UPRT, indicating that engineered UPRT expression will allow cell-specific analysis of gene expression in organisms other than T. gondii. This SuperSeries is composed of the SubSeries listed below.
2006-03-08 | GSE4401 | GEO
Project description:To identify cell type-specific RNA without cell sorting, mice expressing UPRT in a specific cell type were exposed to 4-thiouracil to label RNA only in the UPRT-expressing cells. Total RNA was extracted from tissues and treated with iodoacetamide followed by RNA-seq library preparation. The reads generated from 4-thiouracil-containing transcripts should contain T to C mismatches as a result of 4-thiouracil incorporations and thus can be identified as the transcripts generated in the UPRT-expressing cells.
2018-07-09 | E-MTAB-6353 | biostudies-arrayexpress
Project description:Many stem cell populations exist in a quiescent state in vivo, exiting quiescence and entering the cell cycle in response to specific stimuli. In the case of skeletal muscle, the muscle stem cells (MuSCs, or “satellite cells”) are quiescent under normal homeostatic conditions and undergo activation and cell cycle entry in response to muscle fiber damage. Quiescent MuSCs are also much more potent than their proliferating progeny in assays of stem cell transplantation. In recent years, it has become increasingly apparent that the quiescent state is both actively maintained and dynamically regulated. However, most of the analyses of quiescent MuSCs have come from cells that have been removed from their niche in vivo, purified by fluorescence activated cell sorting, and then assay ex vivo. Although such cells are still in the quiescent state under these conditions, there is no doubt that significant biochemical changes will occur during the isolation and purification process. Thus, we have sought to examine the true in vivo quiescent state by analyzing the transcriptome of MuSCs. To achieve that, we have used techniques to label actively transcribing RNA in vivo using nucleoside analogs. In mice in which the enzyme uracil phosphoribosyltransferase (UPRT) is expressed specifically in MuSCs, administration of 4-thiouracil (4TU), which is converted to thiouridine (TU) by UPRT, resulted in labelling of MuSC transcripts, and the transcriptome could be analyzed following pull-down of TU-tagged RNA. Varying the timing of 4TU administration revealed the dynamic regulation of different subsets of transcripts. Notably, labeling transcripts during the isolation procedure revealed very active transcription of specific subsets of genes. Nevertheless, the ex vivo transcriptome remained largely reflective of the in vivo transcriptome. Using the transcriptional inhibitor, α-amanitin, we were also able to show that there was little difference between the steady-state transcript levels of the most highly expressed genes when comparing the ex vivo transcriptome with the in vivo transcriptome. Together, these data provide a novel view of the molecular regulation of the quiescent state at the transcriptional level, demonstrate the utility of these tools for probing transcriptional dynamics in vivo, and provide an invaluable resource for understanding stem cell state transitions.
2017-11-12 | GSE97399 | GEO