2025-03-15
ENA first public
ERC000011
ENA-CHECKLIST
German Center for Neurodegenerative Diseases
INSDC center name
public
INSDC status
ERS23780547
SRA accession
E-MTAB-14889:Sample 8
Submitter Id
16
month
age
ArrayExpress
broker name
Neurons, Glial cell, Vascular cell
cell_type
not collected
collection date
house mouse
common name
Protocols: 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. 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. 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.
description
human a-syn no overexpression
disease
not collected
geographic location (country and/or sea)
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
brain
organism part
Mus musculus
scientific_name
male, female
sex
Sample 8
title