2024-07-30
ENA first public
ERC000011
ENA-CHECKLIST
Centre for Discovery Brain Sciences, University of Edinburgh
INSDC center name
public
INSDC status
ERS18536122
SRA accession
E-MTAB-13937:MH1202
Submitter Id
12
month
age
ArrayExpress
broker name
astrocyte
cell_type
not collected
collection date
house mouse
common name
Protocols: Mice (3 per age/genotype combination) were anesthetised using isoflurane and decapitated. CO2 was not used in order to avoid confounding effects on gene expression. The neocortices were dissected and placed in ice cold Dulbecco's phosphate-buffered saline (DPBS) (ThermoFisher). Tissue was enzymatically and mechanically dissociated using the Adult Brain Tissue Dissociation Kit, as per the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, tissue was combined with the enzyme cocktails. The gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec) was used to mechanically dissociate the tissue at 37oC for 30 minutes (program 37C_ABDK_01). The digested tissue was filtered (70 μm) with DPBS. Myelin and cell debris were removed using the Debris Removal Solution and centrifugation steps. Erythrocytes were removed using the Red Blood Cell Removal Solution and centrifugation steps. Cells were transported to the FACS facility in PBS containing 0.5% bovine serum albumin. FACS of astrocytes was carried out by the University of Edinburgh FACS facility on a BD Aria II. Gates were set up to gate out debris and dead cells, along with clumps and auto fluorescent cells. GFP positive cells were collected. 10x Genomics sample preparation was done using the 10x Genomics Single Cell 3’ Reagent kits V3 (10x Genomics, Pleasanton, California) according to the manufacturer’s instructions. Incubation of the GEMs produces barcoded cDNA from poly-adenylated mRNA. GEMs were then broken and pooled fractions are recovered. Silane magnetic beads were used to purify the first-strand cDNA from the post GEM-RT reaction mixture, which includes leftover biochemical reagents and primers. Barcoded, full-length cDNA was amplified via PCR to generate sufficient mass for library construction.
description
adult
developmental stage
APP PS1
genotype
not collected
geographic location (country and/or sea)
pooled from three animals
individual
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
cerebral cortex
organism part
Mus musculus
scientific_name
pooled from mixed sex animals
sex
C57BL/6J
strain
MH1202
title