Next generation sequencing of distal colon glial cells with DNBS-induced inflammation and neurokinin-2 receptor antagonism utilizing RiboTag mice
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ABSTRACT:
PROVIDER: PRJNA472607 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7203860_1.fastq.gz | Fastqsanger.gz | |||
| SRR7203860_2.fastq.gz | Fastqsanger.gz | |||
| SRR7203861_1.fastq.gz | Fastqsanger.gz | |||
| SRR7203861_2.fastq.gz | Fastqsanger.gz | |||
| SRR7203862_1.fastq.gz | Fastqsanger.gz |
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Project description:Purpose:We have the first-reported set of glial-specific transcripts utilizing the Ribotag model. We use this model to explore glial changes in DNBS-induced inflammation and neurokinin-2 receptor (NK2R) antagonism. Methods: Actively translated mRNA profiles of the distal colon myeneteric plexi of Rpl22(+/-)Sox10(+/-) male and female mice 8-10 weeks old were obtained utilizing the HA-tagged ribosomal immunoprecipitation and downstream RNA extraction. Samples meeting RNA quality standards by 18S and 28S rRNA peaks by 2100 Bioanalyzer and RNA 6000 Nano LabChip Kit (Agilent) were deep sequenced with the Illumina HiSeq 4000. Results: We mapped approximately 30-50 millions reads per sample to the mouse genome (v88) and identified approximately 100K ribosome-associated transcripts, with Tuxedo workflow, in distal colon glial cells with DNBS-induced inflammation and NK2R antagonism and their respective controls. Of these transcripts, changes in biological processes associated with inflammation and other important enteric nervous system communications between samples have been identified. Conclusions: Our study demonstrates the first use of the Ribotag model to provide glial cell-specific actively-translated mRNA changes in DNBS-induced inflammation with and without functional NK2R signalling.
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Project description:Analysis of Sertoli and Leydig cell “translatome” utilizing an in vivo ribosome tagging strategy (RiboTag) that allows a detailed and physiologically relevant characterization of the polysome-associated mRNAs in vivo. Although progress has been made in the identification of specific transcripts that are translated in Sertoli and Leydig cells and their response to hormones, efforts to expand these studies have been restricted by technical hurdles. Our analysis identified all previously characterized Leydig and Sertoli cell-specific markers and identified in a comprehensive manner novel markers of Leydig and Sertoli cells; the translational response of these two cell types to gonadotropins or testosterone was also investigated. Leydig cell-specific (Cyp17iCre: RiboTag) and Sertoli cell-specific (AMH-Cre: RiboTag) RiboTag mice were obtained by crossing RiboTag homozygous mice with Cyp17iCre or AMH-Cre mice. For in vivo LH treatment experiments, mice were injected with the GnRH antagonist acyline for 4 days before a single injection of purified human LH. After treatment, testes were homogenized and polysomes were immunoprecipitated by utilizing an anti-HA antibody. RNA was extracted, labelled, and hybridized to Mouse Gene ST 1.0 arrays.
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Project description:Analysis of Sertoli and Leydig cell “translatome” utilizing an in vivo ribosome tagging strategy (RiboTag) that allows a detailed and physiologically relevant characterization of the polysome-associated mRNAs in vivo. Although progress has been made in the identification of specific transcripts that are translated in Sertoli and Leydig cells and their response to hormones, efforts to expand these studies have been restricted by technical hurdles. Our analysis identified all previously characterized Leydig and Sertoli cell-specific markers and identified in a comprehensive manner novel markers of Leydig and Sertoli cells; the translational response of these two cell types to gonadotropins or testosterone was also investigated.
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Project description:Transcriptome profiling is widely used to infer functional states of specific cell types, as well as their responses to stimuli, to define contributions to physiology and pathophysiology. Focusing on microglia, the brain macrophages, we report here a side-by-side comparison of classical cell sort-based transcriptome sequencing and the ‘RiboTag’ method that avoids cell retrieval from tissue context and yields translatome sequencing information. Conventional whole cell microglia transcriptomes were found to be significantly tainted by artifacts induced by tissue-dissociation, cargo contaminations and transcripts sequestered from ribosomes. Conversely, our data highlight the added value of RiboTag profiling to assess the accuracy of Cre transgenic mice. Collectively, this study indicates method-based biases, reveals observer effects and establishes RiboTag-based translatome profiling as a valuable complement to standard sort-based profiling strategies.
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Project description:Genetic studies have suggested a role for glial pathology in the genesis of schizophrenia (SCZ). To assess the nature of SCZ-associated human glial dysfunction in vivo, we established human glial chimeric mice using glial progenitor cells (GPCs) produced from induced pluripotential cells (hiPSCs), derived from patients with juvenile-onset schizophrenia or healthy controls. To this end, hiPSC GPCs were implanted neonatally into either immunodeficient myelin wild-type mice, in which donor GPCs remained as progenitors or became astrocytes, or into myelin-deficient shiverer mice, in which the GPCs also gave rise to oligodendrocytes. When implanted into shiverers, the SCZ-derived GPCs exhibited less expansion in the white matter than did control GPCs, instead migrating prematurely into the cortex. The SCZ GPC-transplanted shiverers were consequently hypomyelinated relative to control GPC-engrafted mice. When established instead in myelin wild-type hosts, the SCZ hiPSC glial chimeras manifested markedly delayed and diminished astrocytic differentiation, which was associated with diminished prepulse inhibition and an aberrant behavioral phenotype across multiple modalities. Accordingly, RNA-seq revealed significant differences in both glial differentiation-associated and synaptic gene expression by SCZ GPCs. These data suggest a potent contribution of cell-autonomous glial dysfunction to the development of schizophrenia, and provide a model for the in vivo assessment of human glial pathology in this disorder.
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