2025-06-01
ENA first public
ERC000011
ENA-CHECKLIST
Deutsches Zentrum für Neurodegenerative Krankheiten; German Center for Neurodegenerative Diseases
INSDC center name
public
INSDC status
ERS24611860
SRA accession
E-MTAB-15158:Sample 3
Submitter Id
23
month
age
ArrayExpress
broker name
Neuron, Glial, Vascular
cell type
not collected
collection date
house mouse
common name
Protocols: 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.  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.  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.
description
adult
developmental stage
Control
disease
not collected
geographic location (country and/or sea)
not applicable
isolate
Mus musculus
organism
Mouse
organism
brain
organism part
Mus musculus
scientific_name
male
sex
Sample 3
title