To investigate 4-TU labelling kinetics in a novel zebrafish transgenic line (lf:UPRT). [Adult SLAM-Itseq]
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ABSTRACT:
ORGANISM(S): Danio rerio
PROVIDER: GSE230830 | GEO | 2024/01/13
REPOSITORIES: GEO
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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: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:We investigated the immediate transcriptional changes in hepatocytes following APAP-induced acute liver injury by exposing lf:UPRT zebrafish larvae to DMSO or APAP in the presence of 4TU. We the performed gene expression profiling analysis on total and nascent counts using data obtained from SLAM-ITseq of WT and Nrf2KO animals.
2024-01-13 | GSE230831 | GEO
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.
2009-04-24 | GSE15445 | GEO
Project description:To adapt SLAM-ITseq for Caenorhabditis elegans, we inserted a codon-optimized version of the Toxoplasma gondii UPRT gene downstream of a FRT-flanked mCh::his-58 cassette and under control of the strong and ubiquitous eft-3 promoter. We introduced a single copy of this construct into the C. elegans genome and crossed the resulting line with a dpy-7p::FLP driver. FLP-dependent expression of UPRT was confirmed by Western blotting (strain BN1190). As a proof of concept, we exposed animals to 4-TU from L3 to L4 stage and purified total RNA. RNA from animals without FLP expression was used to control for UPRT-independent labeling strain BN1158). RNA was alkylated with iodoacetamide, which leads to a T>C conversion of 4-TU-labeled positions during cDNA synthesis, and subjected to deep sequencing (see Materials and Methods). We confirmed that the presence of FLP and UPRT did not affect gene expression. Comparing the T>C conversion rate per gene revealed a significant increase in nematodes expressing UPRT in the hypodermis. Next, we performed a beta-binominal test to identify genes that were significantly labelled across all replicas, which retrieved 197 genes (p<0.003; FDR<0.1 after adjustment for multiple comparisons). Original SLAM-ITseq reference: Matsushima W, Herzog VA, Neumann T, Gapp K, Zuber J, Ameres SL, Miska EA (2018) SLAM-ITseq: sequencing cell type-specific transcriptomes without cell sorting. Development 145 (13). doi:10.1242/dev.164640
2022-12-31 | GSE192419 | GEO
Project description:Using a novel method for metabolic labelling of RNA called iTag RNA we isolate hepatocytes' and kidney proximal epithelial cells' transcriptional response to a dietary challenge and identify multiple hepatocyte-secreted small ccfRNAs in plasma, and transfer of these hepatic derived ccfRNAs to VsWAT.
2020-03-02 | GSE143562 | GEO
Project description:Using a novel method for metabolic labelling of RNA called iTag RNA we isolate hepatocytes' and kidney proximal epithelial cells' transcriptional response to a dietary challenge and identify multiple hepatocyte-secreted small ccfRNAs in plasma, and transfer of these hepatic derived ccfRNAs to VsWAT
2022-06-27 | GSE133458 | GEO
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
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:Streptomyces violaceusniger Tu 4113 genome sequencing project
| PRJNA43031 | ENA