{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Jaehyun Lee"],"organism":["Mus musculus"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-17455"],"description":["Using 6-months, 16-months, and 24-months old mice of a inducible expression of human a-syn constructs based Parkinson mouse model, we produced a single nucleus RNA dataset by cutting 0mm Bregma to -5mm Bregma. The Chromium 3’ Single Cell Library Kit (10x Genomics) was used and Sequencing was performed on a NovaSeq 6000. Conditions are included that got pharmacological inhibition of Cdc42 activity.  Processed files can be found on Zenodo: 10.5281/zenodo.21621060"],"repository":["biostudies-arrayexpress"],"sample_protocol":["Sample Treatment - CASIN (Xcess Biosciences M60040) was freshly prepared weekly. A 100 mM dimethyl sulfoxide (DMSO) stock was diluted in (2-Hydroxypropyl)-beta-cyclodextrin (Sigma H5784). Mice received intraperitoneal injections of 25 mg/kg once daily, around midday, for four consecutive days, followed by a one-week break. This cycle was repeated. Treatment started at 12 or 20 M of age. Control animals received vehicle only. A single batch of lyophilized CASIN was used throughout the study.","Sequencing - The Chromium 3’ Single Cell Library Kit (10x Genomics) was used to generate the Gel Beads-In-Emulsion (GEMs), following the guidelines provided by the manufacturer. Shortly, 22,000 of the isolated nuclei from mouse brain tissue were introduced into barcoded Gel Beads using the Chromium Controller. Following GEM-RT incubation, cDNA samples underwent recovery, purification and amplification through a cDNA amplification reaction. Quality assessments on the amplified cDNA were conducted using a High Sensitivity DNA Kit (Agilent) on a TapeStation platform. Libraries were subsequently generated through the processes of fragmentation and adaptor ligation. Sample Index PCR was executed and the resulting purified libraries underwent assessment on TapeStation using a High Sensitivity DNA Kit to evaluate fragment quality. The single-nucleus libraries were then sent to Novogene (United Kingdom) for sequencing with the NovaSeq 6000.","Library Construction - The Chromium 3’ Single Cell Library Kit (10x Genomics) was used to generate the Gel Beads-In-Emulsion (GEMs), following the guidelines provided by the manufacturer. Shortly, 22,000 of the isolated nuclei from mouse brain tissue were introduced into barcoded Gel Beads using the Chromium Controller. Following GEM-RT incubation, cDNA samples underwent recovery, purification and amplification through a cDNA amplification reaction. Quality assessments on the amplified cDNA were conducted using a High Sensitivity DNA Kit (Agilent) on a TapeStation platform. Libraries were subsequently generated through the processes of fragmentation and adaptor ligation. Sample Index PCR was executed and the resulting purified libraries underwent assessment on TapeStation using a High Sensitivity DNA Kit to evaluate fragment quality. The single-nucleus libraries were then sent to Novogene (United Kingdom) for sequencing with the NovaSeq 6000.","Sample Collection - For nuclei isolation from mouse brain tissue, around 100 mg of brain tissue was cut from one hemisphere including 0 mm Bregma to -5 mm Bregma using a mouse coronal brain matrix (Cell Point Scientific). Two of those brain pieces from two different mice were pooled for the extraction. All procedures were carried out on ice. 1400 µl of homogenization buffer (320 mM Sucrose, 5 mM Cacl2, 3 mM Mg(Ac)2, 10 mM Tris HCl pH 8, 0.1 mM EDTA pH 8, 0.1 % NP-40, 1 mM β-mercaptoethanol, 0.4 U/µl RiboLock in H2O) was added to a douncer including the brain tissue and mechanical disruption with around 40 strokes was performed using pestle A followed by pestle B. The resulting homogenate was then passed through a 70 µm Flowmi cell strainer, followed by a 40 µm Flowmi filter. Next, 700 µl of the homogenized suspension was mixed with 450 µl of the working solution (50 % Opti-Prep, 5 mM CaCl2, 3 mM Mg(Ac)2, 10 mM Tris HCl pH 8, 0.1 mM EDTA, 1 mM β-mercaptoethanol in H2O). A gradient was prepared using the following components: 300 µl of 40 % Opti-Prep (40 % Opti-Prep, 96 mM Sucrose, 5 mM CaCl2, 3 mM Mg(Ac)2, 10 mM Tris HCl pH 8, 0.1 mM EDTA, 0.03 % NP-40, 0.12 U/µl RiboLock in H2O), 750 µl of 30 % Opti-Prep (30 % Opti-Prep, 134 mM Sucrose, 5 mM CaCl2, 3 mM Mg(Ac)2, 10 mM Tris HCl pH 8, 0.1 mM EDTA pH 8, 1 mM β-mercaptoethanol, 0.04 % NP-40, 0.17 U/µl RiboLock in H2O) and 700 µl of the mixture of homogenized tissue in working solution. The gradient underwent centrifugation at 10,000 g for 5 min at 4 °C. Following centrifugation, approximately 200 µl of the nuclei were aspirated and transferred into a 1.5 ml low-DNA-binding tube. To this, 250 µl of 2 % BSA and 0.12 U/µl RiboLock in PBS was added. Subsequent centrifugation at 2,000 g for 3 min at 4 °C was performed and the supernatant was discarded. The resulting pellet was resuspended in 250 µl of 2 % BSA and 0.12 U/µl RiboLock, followed by a second round of centrifugation at 2,000 g for 3 min at 4 °C. After discarding the supernatant, the pellet was once again resuspended in 250 µl of 2 % BSA and 0.12 U/µl RiboLock and filtered through a 40 µm Flowmi cell strainer into a low-DNA-binding tube. Further centrifugation at 2,000 g for 3 min at 4 °C was carried out. The pellet obtained was then resuspended in 50 µl of 1x nuclei buffer (1x nuclei buffer of 10x Genomics, with 1 mM DTT, 1 U/µl RiboLock in H2O). For counting nuclei and quality check for nuclear membrane integrity, nuclei were stained with DAPI. The final nuclei were then directly used for the Single-nuclei RNA isolation protocol.","Nucleic Acid Extraction - For nuclei isolation from mouse brain tissue, around 100 mg of brain tissue was cut from one hemisphere including 0 mm Bregma to -5 mm Bregma using a mouse coronal brain matrix (Cell Point Scientific). Two of those brain pieces from two different mice were pooled for the extraction. All procedures were carried out on ice. 1400 µl of homogenization buffer (320 mM Sucrose, 5 mM Cacl2, 3 mM Mg(Ac)2, 10 mM Tris HCl pH 8, 0.1 mM EDTA pH 8, 0.1 % NP-40, 1 mM β-mercaptoethanol, 0.4 U/µl RiboLock in H2O) was added to a douncer including the brain tissue and mechanical disruption with around 40 strokes was performed using pestle A followed by pestle B. The resulting homogenate was then passed through a 70 µm Flowmi cell strainer, followed by a 40 µm Flowmi filter. Next, 700 µl of the homogenized suspension was mixed with 450 µl of the working solution (50 % Opti-Prep, 5 mM CaCl2, 3 mM Mg(Ac)2, 10 mM Tris HCl pH 8, 0.1 mM EDTA, 1 mM β-mercaptoethanol in H2O). A gradient was prepared using the following components: 300 µl of 40 % Opti-Prep (40 % Opti-Prep, 96 mM Sucrose, 5 mM CaCl2, 3 mM Mg(Ac)2, 10 mM Tris HCl pH 8, 0.1 mM EDTA, 0.03 % NP-40, 0.12 U/µl RiboLock in H2O), 750 µl of 30 % Opti-Prep (30 % Opti-Prep, 134 mM Sucrose, 5 mM CaCl2, 3 mM Mg(Ac)2, 10 mM Tris HCl pH 8, 0.1 mM EDTA pH 8, 1 mM β-mercaptoethanol, 0.04 % NP-40, 0.17 U/µl RiboLock in H2O) and 700 µl of the mixture of homogenized tissue in working solution. The gradient underwent centrifugation at 10,000 g for 5 min at 4 °C. Following centrifugation, approximately 200 µl of the nuclei were aspirated and transferred into a 1.5 ml low-DNA-binding tube. To this, 250 µl of 2 % BSA and 0.12 U/µl RiboLock in PBS was added. Subsequent centrifugation at 2,000 g for 3 min at 4 °C was performed and the supernatant was discarded. The resulting pellet was resuspended in 250 µl of 2 % BSA and 0.12 U/µl RiboLock, followed by a second round of centrifugation at 2,000 g for 3 min at 4 °C. After discarding the supernatant, the pellet was once again resuspended in 250 µl of 2 % BSA and 0.12 U/µl RiboLock and filtered through a 40 µm Flowmi cell strainer into a low-DNA-binding tube. Further centrifugation at 2,000 g for 3 min at 4 °C was carried out. The pellet obtained was then resuspended in 50 µl of 1x nuclei buffer (1x nuclei buffer of 10x Genomics, with 1 mM DTT, 1 U/µl RiboLock in H2O). For counting nuclei and quality check for nuclear membrane integrity, nuclei were stained with DAPI. The final nuclei were then directly used for the Single-nuclei RNA isolation protocol."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","MAGE-TAB Files"],"data_protocol":["Data Transformation - Sequencing and alignment of single nucleus samples Paired 150bp snRNA-seq was performed using the 10X Genomics Gene Expression (GEX) 3´protocol with an Novaseq 6000 sequencer. For the alignment of reads, a custom reference was created by adding the sequences of the S1/S2 transgene and the CamkIIa promoter to the mm10 mouse reference genome. Count matrices were obtained using the cellranger count 7.1 pipeline, including introns. Six samples were mapped using the bwUni2.0 High-Performance Computing infrastructure. Quality Control and Integration of cells and counts The unfiltered count matrices were loaded into R and corrected for ambient mRNA using SoupX 1.6.0 with default settings, adjusting “tfidfMin” settings between 0.9 and 1.3 depending on the sample. Seurat objects were created for each sample and subsequently merged. Cells were filtered out based on the following criteria: number of unique molecular identifiers (nUMI) < 2500, number of genes (nGene) < 1500, mitochondrial gene percentage > 3%, ribosomal gene percentage > 1.5%, or log10(Genes/nUMI) < 0.85. Subsequently, doublets and sex-doublets were removed using scDblFinder 1.16.0 and cellXY 0.99.0. Normalization was performed using the SCTransform function on 4000 variable features with glmGamPoi method implemented in Seurat 5.0.1, and top 50 embeddings were obtained via scVI (scvi-tools 1.1.1) integration for sex, age, batch, and number of pooled animals. Clustering was done using the Leiden algorithm and visualized with Uniform Manifold Approximation and Projection algorithm (UMAP). Clusters represented by few samples, less than 100 cells, or a single batch, and not of conditions of interest, were removed. Clusters driven by ribosomal or mitochondrial genes, as well as markers of hindbrain and olfactory cell types, were also discarded. The steps from normalization onward were repeated until no further clusters needed removal. Final integration was performed using harmony 1.2.0 with an integration diversity penalty (theta) of 2, followed by final clustering based on the top 30 harmony components and UMAP visualization. Each subsequent clustering for annotation of sub-cell types was computed following the same procedure. Annotation of Cell types Clusters were annotated in a hierarchical manner using literature, the Mouse Brain Atlas (mousebrain.org), the Human Protein Atlas, and markers identified via the FindConservedMarkers function in Seurat. First, neurons and non-neuronal cells were distinguished using mainly canonical markers, such as but not limited to Rbfox3 (neurons), Mbp (oligodendrocytes), Acsbg1 (astrocytes), Pdgfra (oligodendrocyte precursor cells), Cx3cr1 (microglia), Colec12 (vascular cells), and Ttr (choroid plexus cells). Neurons were further classified into Vglut1, Vglut2, GABA, cholinergic, and dopaminergic neurons. Vglut1 and GABA neurons were further sub-annotated."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina NovaSeq 6000"],"study_type":["single nucleus RNA sequencing"],"species":["Mus musculus"],"pubmed_authors":["Karin Danzer","Jaehyun Lee"],"additional_accession":[]},"is_claimable":false,"name":"snRNA data of Brain section from Parkinson Mouse Model based on inducible expression of human a-syn constructs including conditions with pharmacological CDC42 inhibition","description":"Using 6-months, 16-months, and 24-months old mice of a inducible expression of human a-syn constructs based Parkinson mouse model, we produced a single nucleus RNA dataset by cutting 0mm Bregma to -5mm Bregma. The Chromium 3’ Single Cell Library Kit (10x Genomics) was used and Sequencing was performed on a NovaSeq 6000. Conditions are included that got pharmacological inhibition of Cdc42 activity.  Processed files can be found on Zenodo: 10.5281/zenodo.21621060","dates":{"release":"2026-08-14T00:00:00Z","modification":"2026-08-14T01:00:45.374Z","creation":"2026-08-04T11:38:09.633Z"},"accession":"E-MTAB-17455","cross_references":{"ENA":["ERP203282"],"Biostudies":["E-MTAB-14889"],"EFO":["EFO_0002944","EFO_0004170","EFO_0009809","EFO_0005518","EFO_0003816","EFO_0004184","EFO_0003969"]}}