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| Status |
Public on Nov 18, 2024 |
| Title |
day 3 [scRNA-Seq] |
| Sample type |
SRA |
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| Source name |
brain
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| Organism |
Mus musculus |
| Characteristics |
tissue: brain
strain: C57Black/6
age: 3 month
Sex: male
condition: 3 days
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| Treatment protocol |
Prior to the induction of MCAO, mice were anaesthetized with 3% isoflurane (Aerrane, Baxter) and amaintained in 2% isoflurane using a vaporizer (Tec-3, Cyprane Ltd.). A skin incision between the orbit and the external auditory meatus was made, and a 1-2 mm hole was drilled through the frontal bone 1 mm rostral to the fusion of the zygoma and the squamosal bone, about 3.5 mm ventrally to the dorsal surface of the brain. The middle cerebral artery (MCA) was exposed after the dura was opened and removed. The MCA was occluded by short coagulation with bipolar tweezers (SMT) at a proximal location, followed by transection of the vessel to ensure permanent occlusion. During the surgery, body temperature was maintained at 37 ± 1C using a heating pad. This MCAO model yields small infarct lesions in the parietal cortical region. Intact cortical tissue from 3 months old mice was used as control.
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| Extracted molecule |
total RNA |
| Extraction protocol |
The Plp1/tdTomato mice were deeply anesthetized with pentobarbital (PTB) (100 mg/kg, i.p.), and perfused transcardially with a cold (4–8°C) isolation buffer containing (in mM): NaCl 136.0, KCl 5.4, HEPES 10.0, glucose 5.5, osmolality 290±3 mOsmol/kg. To isolate the control and ischemic areas, the brain was sliced into 600 µm coronal sections using a vibrating microtome Leica VT1200S (Leica Microsystems). The collected tissue was incubated with continuous shaking at 37°C for 45 min in 1 ml of papain solution (20 U/ml) and 0.2 ml DNase (both from Worthington) prepared in isolation buffer. After papain treatment, the tissue was mechanically dissociated by gentle trituration using a 1 ml pipette. The dissociated cells were layered on top of 5 ml of ovomucoid inhibitor solution (Worthington) and harvested by centrifugation (70 x g for 6 min). This method routinely yielded ~2 x 10^6 cells per mouse brain. Cell aggregates were removed by filtering with 70 µm cell strainers (Becton Dickinson). Three animals per condition were pooled in the preparation of cell suspension. After obtaining an aliquot for FACS, the cell suspension was spin down, concentrated, and used for library preparation. Single cell suspension from Plp1/tdTomato mice were sorted using fluorescent activated cell sorting (FACS; BD Influx). The flow cytometer was manually calibrated to deposit a single cell in the center of collection tube. Hoechst 33258 (Life Technologies) was added to the suspension of cells to check viability. 3000 Plp1+ cells from each group (control, D1, D3, D7) were collected into 96-well plates (Life Technologies) coated with BSA (ThermoFisher Scientific) and containing 5 μl of DMEM (ThermoFisher Scientific) and 15% FBS (HyClone).
Single-cell RNA-Seq (scRNA-Seq) libraries were prepared using Chromium Next GEM Single Cell 3' Kit v3.1 (1000268, 10X Genomics) according to the manufacturer’s protocol.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
10x Genomics
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| Data processing |
Libraries were controlled for contamination using fastq_screen v0.11.1. Reads were mapped against the mouse genome GRCm38 and counted using annotation genecode.vM8 using STAR v2.7.3a with parameters “--soloType CB_UMI_Simple --soloFeatures Gene –soloCBmatchWLtype 1MM_multi_pseudocounts –soloUMIdedup 1MM_Directional –soloUMIfiltering MultiGeneUMI”. Empty droplets were filtered out using emptyDrops command from DropletUtils package (v 1.14.1) with lower parameter set to 1000.
Assembly: mm10/GRCm38
Supplementary files format and content: bardcodes.tsv: list of cell barcodes
Supplementary files format and content: features.tsv: list of gene Ids
Supplementary files format and content: matrix.mtx: gene expression count data in Matrix Market Exchange Format
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| Submission date |
May 31, 2023 |
| Last update date |
Nov 18, 2024 |
| Contact name |
Lukas Valihrach |
| Organization name |
Institute of Biotechnology, Czech Academy of Science
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| Lab |
Laboratory of Gene Expression
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| Street address |
Prumyslova 595
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| City |
Vestec |
| ZIP/Postal code |
25250 |
| Country |
Czech Republic |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE233812 |
Spatiotemporal transcriptomic map of glial cell response in a mouse model of acute brain ischemia [scRNA-Seq] |
| GSE233815 |
Spatiotemporal transcriptomic map of glial cell response in a mouse model of acute brain ischemia |
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| Relations |
| BioSample |
SAMN35546770 |
| SRA |
SRX20554855 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7437215_sc-D3_barcodes.tsv.gz |
17.5 Mb |
(ftp)(http) |
TSV |
| GSM7437215_sc-D3_genes.tsv.gz |
379.7 Kb |
(ftp)(http) |
TSV |
| GSM7437215_sc-D3_matrix.mtx.gz |
22.5 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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