Sample 11
Mus musculus
Protocols: Extracted mouse brain tissue was stored overnight at 4 °C in Tissue Storage Solution (Miltenyi Biotec, 130-100-008) and dissociated the next day using the Adult Brain Dissociation Kit (Miltenyi Biotec, 130-107-677) per the manufacturer's instructions. Pellets from two animals were pooled and processed immediately for microglia isolation. All subsequent steps used pre-cooled solutions on ice unless stated otherwise. To improve microglia purity, oligodendrocytes were first depleted. Pellets were resuspended in 97.5 μL PB buffer (D-PBS pH 7.2, 0.5% BSA) with 2.5 μL Anti-O4 MicroBeads (Miltenyi Biotec, 130-096-670) and incubated for 15 min at 2–8 °C in the dark. Cells were washed with 1 mL PB buffer (300 × g, 5 min), resuspended in 500 μL PB buffer, and applied to an MS Column fitted with a 70 μm Pre-Separation Filter in a MACS Separator. The flow-through and three subsequent 500 μL PB washes were combined as the unlabeled fraction; the column with labeled oligodendrocytes was discarded. The combined flow-through was centrifuged (300 × g, 10 min, 4 °C), resuspended in 90 μL PB buffer with 10 μL CD11b MicroBeads (Miltenyi Biotec, 130-093-636), and incubated for 15 min at 2–8 °C in the dark. Cells were washed with 1 mL PB buffer (300 × g, 5 min, 4 °C), resuspended in 500 μL PB buffer, and applied to an equilibrated MS Column in the MACS Separator. After three 500 μL PB washes (allowing complete emptying between washes), the column was removed from the separator and magnetically labeled microglia were eluted with 1 mL PB buffer using the plunger. The positive fraction was centrifuged (300 × g, 5 min, 4 °C), resuspended in 350 μL RLT buffer, and stored at –80 °C, yielding ~2 M microglia per sample. RNA was extracted using the Quick-RNA Microprep Kit (Zymo Research, R1051) with on-column DNase I treatment, per the manufacturer's instructions, eluted in DNase/RNase-free water, and stored at −80 °C. RNA quality and quantity were assessed using a TapeStation 4150 system (Agilent Technologies) with RNA ScreenTape. Strand-specific RNA-seq libraries were prepared using the sparQ RNA-Seq HMR Kit (QuantaBio, 733-2900) with sparQ PureMag Beads (QuantaBio, 733-2626) and sparQ UDI Adapters (QuantaBio, 95211-096) according to the manufacturer's instructions. Amplified libraries were purified by three consecutive clean-up steps using 45 µL sparQ PureMag Beads per purification (0.9× bead-to-sample ratio). Final library quality and concentration were assessed on a TapeStation 4150 system (Agilent Technologies) with D1000 ScreenTape.
ENA first public
2026-06-04
INSDC center name
Deutsches Zentrum für Neurodegenerative Krankheiten; German Center for Neurodegenerative Diseases
INSDC status
public
Submitter Id
E-MTAB-17136:Sample 11
age
8
broker name
ArrayExpress
cell type
microglial cell
collection date
not collected
common name
house mouse
developmental stage
adult
disease
Parkinson's disease
geographic location (country and/or sea)
not collected
isolate
not applicable
organism part
brain
sample name
E-MTAB-17136:Sample 11
scientific_name
Mus musculus
sex
female