2024-08-22
ENA first public
ERC000011
ENA-CHECKLIST
Centre for Discovery Brain Sciences, University of Edinburgh
INSDC center name
public
INSDC status
ERS20223358
SRA accession
E-MTAB-14160:1825
Submitter Id
14
day
age
ArrayExpress
broker name
all cells
cell_type
not collected
collection date
house mouse
common name
Protocols: To sort astrocytes, mouse neocortices were rapidly harvested then dissociated with Adult Brain Dissociation Kit (Miltenyi Biotec) on a gentleMACS Octo Dissociator using program 37C_ABDK_01. Dissociated samples were then treated with debris and red blood cell removal steps to obtain cell suspensions. For FACS, cells were resuspended in a final volume of 100 uL 0.1% PB buffer. Then cells were incubated with fluorescent conjugated antibodies at 4° C for 30 minutes. The following antibodies were used: ACSA2 APC (1:200, Miltenyi Biotec, 130-116-245) and O4 PE (1:100, Miltenyi Biotec, 30-117-357). ACSA2 APC (+) and O4-PE (-) live single cells were selected as the astrocyte population.   For single nucleus-RNAseq, neocortices were acutely collected from Csf1rΔFIRE/ΔFIRE (n=6) and wildtype (n=6), flash-frozen, and then stored at -80°C. Nuclei isolation kit (Sigma-Aldrich; NUC201-1KT), DTT (R0861), RNAse inhibitor (Life Technologies; AM2694), 30 µm cell strainer (Partec CellTrics), and 30 G insulin needles (BD; 324826) were used. Cryopreserved tissue samples were thawed on ice. Lysis was performed with PURE buffer, 0.1M DTT, 10% Triton, and RNAse inhibitor. The samples were mechanically dissociated in lysis buffer using a P-1000 pipette 10 times, and further 3 times with insulin needles. The lysate was mixed with sucrose cushion, which was prepared using PURE 2M sucrose, sucrose cushion buffer, 0.1M DTT, and RNAse inhibitor. This mixture was then filtered through a 30 µm cell strainer. In a new low-binding tube, 200 µl of sucrose cushion was added and carefully overlaid with 560 µl of the filtrate. The samples were centrifuged for 45 minutes at full speed, at 4°C. The supernatant was discarded, and the pellet was resuspended in Dulbecco's phosphate-buffered saline (DPBS) (14190-094) + 0.5% BSA with RNAse inhibitor. The suspension was then centrifuged at 1000 G, repeated, and finally resuspended in 200 µl DPBS + 0.5% BSA with RNAse inhibitor before proceeding to FANS. Sorting of nuclei was done by flow cytometry was performed using a BD FACSAria II cell sorter (BD Biosciences). The gating was set based on size and granularity using Forward Scatter (FSC) and Side Scatter (SSC) to capture singlets and remove debris. Nuclei were stained with DAPI for detection. Each sample was sorted into separate tubes, with a total of 100,000 particles per tube. Quality control was conducted using Luna FX7 with Acridine Orange/Propidium Iodide (AO/PI), and the input nuclei number was 20,000 per sample. Library preparation were conducted with the Chromium Single Cell 3′ v3.1 Reagent Kit (10x Genomics), following the manufacturer’s instructions. library preparation were conducted with the Chromium Single Cell 3′ v3.1 Reagent Kit (10x Genomics), following the manufacturer’s instructions.
description
juvenile stage
developmental stage
Csf1r ∆FIRE/∆FIRE
genotype
not collected
geographic location (country and/or sea)
1825
individual
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
neocortex
organism part
Mus musculus
scientific_name
female
sex
C57BL/6J / CBA mixed background
strain
1825
title