biostudies-arrayexpressUnknownTranscriptomicsGenomicsProteomicsJaehyun LeeIllumina NovaSeq 6000RNA-seq of coding RNA from single cellsMouseMousehttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15158Using 23-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. Processed count matrices, metadata and objects with filtered, clustered, and annotated data can be found on Zenodo: 10.5281/zenodo.14988055biostudies-arrayexpressLibrary Construction - To isolate nuclei from murine brain tissue, approximately 100 mg of brain material was dissected from a single hemisphere, spanning the region from 0 to -5 mm relative to Bregma, using a mouse coronal brain matrix (Cell Point Scientific). Brain tissue from two animals was pooled to ensure sufficient material for subsequent isolation procedures. All experimental steps were carried out on ice to maintain sample integrity. Initially, 1,400 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 % Nonidet P40 Substituent (NP-40), 1 mM β-mercaptoethanol, 0.4 U/µl RiboLock in H2O) was added to a douncer containing the dissected brain tissue. The tissue was mechanically homogenized using pestle A, followed by further homogenization with pestle B. The resulting homogenate was filtered sequentially through 70 µm and 40 µm Flowmi cell strainers to eliminate debris and large particles. Subsequently, 700 µl of the filtered homogenate was combined with 450 µl of a 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 density gradient was prepared using 300 µl of 40 % Opti-Prep (with 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 (with 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 homogenized tissue mixture. The density gradient was centrifuged at 10,000 g for 5 min at 4 °C. Following centrifugation, 200 µl of the isolated nuclei were carefully aspirated and transferred into a 1.5 ml low-DNA-binding tube. To preserve nuclear integrity, 250 µl of a solution containing 2 % BSA and 0.12 U/µl RiboLock in PBS was added. The mixture was centrifuged at 2,000 g for 3 min at 4 °C, and the supernatant was discarded. The resulting pellet was resuspended in 250 µl of the same 2 % BSA/RiboLock solution and subjected to a second round of centrifugation under identical conditions. The supernatant was discarded, and the pellet was resuspended in 250 µl of 2 % BSA/RiboLock solution. The suspension was filtered through a 40 µm Flowmi cell strainer into a fresh low-DNA binding tube. A final centrifugation at 2,000 g for 3 min at 4 °C was performed to pellet the nuclei. The resulting pellet was resuspended in 50 µl of 1x nuclei buffer, prepared by diluting 10X Genomics nuclei buffer with the addition of 1 mM dithiothreitol (DTT) and 1 U/µl RiboLock in H2O. To evaluate the integrity and quantity of the isolated nuclei, a DAPI stain was applied to visualize nuclear membrane integrity. The isolated nuclei were subsequently used for snRNA-seq experiments.Sequencing - The “Chromium 3’ Single Cell Gene Expression and 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.Nucleic Acid Extraction - To isolate nuclei from murine brain tissue, approximately 100 mg of brain material was dissected from a single hemisphere, spanning the region from 0 to -5 mm relative to Bregma, using a mouse coronal brain matrix (Cell Point Scientific). Brain tissue from two animals was pooled to ensure sufficient material for subsequent isolation procedures. All experimental steps were carried out on ice to maintain sample integrity. Initially, 1,400 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 % Nonidet P40 Substituent (NP-40), 1 mM β-mercaptoethanol, 0.4 U/µl RiboLock in H2O) was added to a douncer containing the dissected brain tissue. The tissue was mechanically homogenized using pestle A, followed by further homogenization with pestle B. The resulting homogenate was filtered sequentially through 70 µm and 40 µm Flowmi cell strainers to eliminate debris and large particles. Subsequently, 700 µl of the filtered homogenate was combined with 450 µl of a 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 density gradient was prepared using 300 µl of 40 % Opti-Prep (with 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 (with 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 homogenized tissue mixture. The density gradient was centrifuged at 10,000 g for 5 min at 4 °C. Following centrifugation, 200 µl of the isolated nuclei were carefully aspirated and transferred into a 1.5 ml low-DNA-binding tube. To preserve nuclear integrity, 250 µl of a solution containing 2 % BSA and 0.12 U/µl RiboLock in PBS was added. The mixture was centrifuged at 2,000 g for 3 min at 4 °C, and the supernatant was discarded. The resulting pellet was resuspended in 250 µl of the same 2 % BSA/RiboLock solution and subjected to a second round of centrifugation under identical conditions. The supernatant was discarded, and the pellet was resuspended in 250 µl of 2 % BSA/RiboLock solution. The suspension was filtered through a 40 µm Flowmi cell strainer into a fresh low-DNA binding tube. A final centrifugation at 2,000 g for 3 min at 4 °C was performed to pellet the nuclei. The resulting pellet was resuspended in 50 µl of 1x nuclei buffer, prepared by diluting 10X Genomics nuclei buffer with the addition of 1 mM dithiothreitol (DTT) and 1 U/µl RiboLock in H2O. To evaluate the integrity and quantity of the isolated nuclei, a DAPI stain was applied to visualize nuclear membrane integrity. The isolated nuclei were subsequently used for snRNA-seq experiments.Sample Collection - To isolate nuclei from murine brain tissue, approximately 100 mg of brain material was dissected from a single hemisphere, spanning the region from 0 to -5 mm relative to Bregma, using a mouse coronal brain matrix (Cell Point Scientific). Brain tissue from two animals was pooled to ensure sufficient material for subsequent isolation procedures. All experimental steps were carried out on ice to maintain sample integrity. Initially, 1,400 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 % Nonidet P40 Substituent (NP-40), 1 mM β-mercaptoethanol, 0.4 U/µl RiboLock in H2O) was added to a douncer containing the dissected brain tissue. The tissue was mechanically homogenized using pestle A, followed by further homogenization with pestle B. The resulting homogenate was filtered sequentially through 70 µm and 40 µm Flowmi cell strainers to eliminate debris and large particles. Subsequently, 700 µl of the filtered homogenate was combined with 450 µl of a 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 density gradient was prepared using 300 µl of 40 % Opti-Prep (with 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 (with 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 homogenized tissue mixture. The density gradient was centrifuged at 10,000 g for 5 min at 4 °C. Following centrifugation, 200 µl of the isolated nuclei were carefully aspirated and transferred into a 1.5 ml low-DNA-binding tube. To preserve nuclear integrity, 250 µl of a solution containing 2 % BSA and 0.12 U/µl RiboLock in PBS was added. The mixture was centrifuged at 2,000 g for 3 min at 4 °C, and the supernatant was discarded. The resulting pellet was resuspended in 250 µl of the same 2 % BSA/RiboLock solution and subjected to a second round of centrifugation under identical conditions. The supernatant was discarded, and the pellet was resuspended in 250 µl of 2 % BSA/RiboLock solution. The suspension was filtered through a 40 µm Flowmi cell strainer into a fresh low-DNA binding tube. A final centrifugation at 2,000 g for 3 min at 4 °C was performed to pellet the nuclei. The resulting pellet was resuspended in 50 µl of 1x nuclei buffer, prepared by diluting 10X Genomics nuclei buffer with the addition of 1 mM dithiothreitol (DTT) and 1 U/µl RiboLock in H2O. To evaluate the integrity and quantity of the isolated nuclei, a DAPI stain was applied to visualize nuclear membrane integrity. The isolated nuclei were subsequently used for snRNA-seq experiments.OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesKarin DanzerJaehyun LeefalsesnRNA Visium Spatial data of Brain section from Parkinson Mouse Model based on inducible expression of human a-syn constructs 23 monthsUsing 23-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. Processed count matrices, metadata and objects with filtered, clustered, and annotated data can be found on Zenodo: 10.5281/zenodo.149880552025-06-01T00:00:00Z2025-05-21T14:06:42.874Z2025-05-21T14:06:42.874ZE-MTAB-15158ERP172806EFO_0002944EFO_0004170EFO_0005684EFO_0005518EFO_0004184