Synaptic small and total RNAome from hippocampal Synaptosomes and the synaptic compartments of microfluidic chambers
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA668132 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12795974.fastq.gz | Fastqsanger.gz | |||
| SRR12795975.fastq.gz | Fastqsanger.gz | |||
| SRR12795976.fastq.gz | Fastqsanger.gz | |||
| SRR12795977.fastq.gz | Fastqsanger.gz | |||
| SRR12795978.fastq.gz | Fastqsanger.gz |
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Project description:Here we isolate high quality synaptosomes from the hippocampus of young wild type mice and provide, at an unprecedented level, the coding and small non-coding synaptic RNAome. These data are complimented by a novel approach to analyze the synaptic RNAome from primary hippocampal neurons grown in microfluidic chambers. Our data show that synaptic microRNAs control almost the entire synaptic mRNAome and we identified several hub microRNAs. By combining the in vivo synaptosomal data with our novel microfluid chamber system, we also provide evidence to support the hypothesis that part of the synaptic microRNAome may be supplied to neurons via astrocytes. Moreover, the microfluidic system is suitable to study the dynamics of the synaptic RNAome in response to stimulation as shown with KCL treatment.
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Project description:Current RNA expression profiling methods rely on enrichment steps for specific RNA classes, thereby not detecting all RNA species in an unperturbed manner. We report strand-specific RNAome sequencing that determines expression of small and large RNAs from ribosomal RNA-depleted total RNA in a single sequence run. Since current analysis pipelines cannot reliably analyze small and large RNAs simultaneously, we developed TRAP, Total Rna Analysis Pipeline, a robust interface that is also compatible with existing RNA sequencing protocols. RNAome sequencing quantitatively preserved all RNA classes, allowing cross-class comparisons that facilitates the identification of relationships between different RNA classes. We demonstrate the strength of RNAome sequencing in mouse embryonic stem cells treated with cisplatin. MicroRNA and mRNA expression in RNAome sequencing significantly correlated between replicates and was in concordance with both existing RNA sequencing methods and gene expression arrays generated from the same samples. Moreover, RNAome sequencing also detected additional RNA classes such as enhancer RNAs, anti-sense RNAs, novel RNA species and numerous differentially expressed RNAs undetectable by other methods. At the level of complete RNA classes, RNAome sequencing also identified a specific global repression of the microRNA and microRNA isoform classes after cisplatin treatment whereas all other classes such as mRNAs were unchanged. These characteristics of RNAome sequencing will significantly improve expression analysis as well as studies on RNA biology not covered by existing methods. 8 mouse embryonic stem cell samples, control and cisplatin treated, 4 replicates per group
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Project description:Current RNA expression profiling methods rely on enrichment steps for specific RNA classes, thereby not detecting all RNA species in an unperturbed manner. We report strand-specific RNAome sequencing that determines expression of small and large RNAs from ribosomal RNA-depleted total RNA in a single sequence run. Since current analysis pipelines cannot reliably analyze small and large RNAs simultaneously, we developed TRAP, Total Rna Analysis Pipeline, a robust interface that is also compatible with existing RNA sequencing protocols. RNAome sequencing quantitatively preserved all RNA classes, allowing cross-class comparisons that facilitates the identification of relationships between different RNA classes. We demonstrate the strength of RNAome sequencing in mouse embryonic stem cells treated with cisplatin. MicroRNA and mRNA expression in RNAome sequencing significantly correlated between replicates and was in concordance with both existing RNA sequencing methods and gene expression arrays generated from the same samples. Moreover, RNAome sequencing also detected additional RNA classes such as enhancer RNAs, anti-sense RNAs, novel RNA species and numerous differentially expressed RNAs undetectable by other methods. At the level of complete RNA classes, RNAome sequencing also identified a specific global repression of the microRNA and microRNA isoform classes after cisplatin treatment whereas all other classes such as mRNAs were unchanged. These characteristics of RNAome sequencing will significantly improve expression analysis as well as studies on RNA biology not covered by existing methods. 8 mouse embryonic stem cell samples, 4 control and 4 cisplatin treated, 4 replicates per group for Affymetrix arrays. Of these, 6 were used for sequencing and are reported in this Series.
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Project description:Preliminary Summary: In this study, we set to specifically analyze effects of aging on differential splicing in the local synaptic proteome. To this end, we analyzed the RNA extracted from synaptosomes obtained from cerebral cortices of C57BL/6 mice of 3 weeks, 5 months and 19 months of age. Synaptosomes are artificial, membranous sacs that contain synaptic components and are generated by subcellular fractionation of homogenized or ground-up nerve tissue. We employ RNA-seq using paired long-reads to obtain comprehensive sequence information on synaptic transcripts and two different bioinformatic methods (DIEGO and LeafCutter) to quantify differential splicing. Finally, the change in the usage of the selected junctions was validated through RT-qPCR and RT-PCR using an independent set of samples.
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2015-04-01 | GSE48083 | GEO
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Project description:current synapse proteomics are restricted to “average” composition of abundant synaptic proteins. Here we performed a subcellular proteomic workflow that could identify and quantify the deep proteome of synaptic vesicles purified from rat whole brain, including previously missing proteins present in a small percentage of central synapses. This synaptic vesicle proteome newly detected many proteins of physiological and pathological relevance particularly in low abundance range, thus providing a resource for future investigations on diversified synaptic functions and neuronal dysfunctions.
Raw data from 3 replicates of synaptosomes (P2') and 3 replicates of SV are deposited here. Each replicate coming from 24 fractions from a peptide fractionation step in the workflow.
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Project description:Cystatin B (CSTB) is a small protease inhibitor involved in cell proliferation and migration, and composition of extracellular matrix during brain development. Loss-of-function mutations in the gene encoding CSTB cause progressive myoclonic epilepsy 1 (EPM1). We previously demonstrated that CSTB is locally synthesized in the synaptosomes from rat brain and secreted into the media, indicating its role in synaptic plasticity. In this work, we further investigate the involvement of CSTB in synaptic plasticity, using synaptosomes from rodents’ brain, as well as from human Cerebral Organoids (hCOs). Our data demonstrated that CSTB is released by synaptosomes in two ways: as a soluble protein, and in extracellular vesicles-mediated manner. Synaptosomes isolated from hCOs are enriched in pre-synaptic proteins, as expected for an in vitro model of human synapses, and contain CSTB at all developmental stages analysed. However, only at late maturation stages synapse becomes physiologically active, i.e. particularly enriched with exocytosis-associated proteins. CSTB presence in the synaptic territories was confirmed also by immunostaining on human neurons in vitro. To investigate if the depletion of CSTB in EPM1 is affecting synaptic plasticity, we used synaptosomes from EPM1 hCOs. The synaptosomal levels of presynaptic proteins and of an initiation factor linked to local protein synthesis, were reduced in EPM1 hCOs compared to controls, and the extracellular vesicles trafficking was impaired. Moreover, morphological alteration was detected in neurons from EPM1 patients, indicating a connectivity deficit. In conclusion, our data indicate a significant role of synaptic CSTB in cell-to-cell communication in physiological and pathological conditions, and demonstrated that CSTB-dependent altered synaptic plasticity is one of the mechanisms underlying EPM1.
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