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| Status |
Public on Jul 02, 2019 |
| Title |
WT Microglia biological replicate 3 |
| Sample type |
RNA |
| |
|
| Source name |
Microglia from ischemic hemisphere of WT mice
|
| Organism |
Mus musculus |
| Characteristics |
genotype: WT
gender: male
strain: C57BL/6
tissue: brain
cell type: microglia
age: 8-10 weeks
|
| Treatment protocol |
8-10 week-old male C57BL/6 mice, of genotype TREM1+/+ and TREM1-/-, underwent MCAo for 45min followed by reperfusion (MCA0-RP). 48 hours after the reperfusion, mice were transcardially perfused with cold heparinized 0.9% NaCl and ischemic hemispheres were homogenized manually to filter through 70µm cell strainer. All cells in the homogenate were then purified by myelin depletion with Miltenyi Myelin removal kit according to the manufacturer’s instructions. After cell staining with CD45, CD11b and Ly6g, neutrophils (CD45Hi, CD11b+, Ly6G+), macrophages (CD45Hi, CD11b+, Ly6G-) and microglia (CD45Lo, CD11b+, Ly6G+) were sorted and RNA isolated for Affymetrix Clariom HT microarray analysis.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA from neutrophils, macrophages and microglia from TREM1 -/- and +/+ ischemic hemispheres was extracted with Qiagen RNeasy Micro kit according to the manufacturer’s instructions.
|
| Label |
biotin
|
| Label protocol |
Microarray was performed at the Protein and Nucleic Acid (PAN) Facility at the Stanford University. The RNA integrity was examined on a bioanalyzer (Agilent). High-quality RNA (RNA integrity number >7.5) was used for expression microarray analysis. 0.6-0.8 ng of total RNA was used to generate double strand cDNA. 6.6 μg of ds cDNA were fragmented and labeled with biotin according to Affymetrix GeneChip Pico Reagent Kit manual (Thermo Fisher).
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| |
|
| Hybridization protocol |
3.0 μg of the fragmented and labeled cDNA was hybridized to Mouse Clariom S array at 60 rpm for 16 h at 45°C in an Affymetrix Hybridization Oven 640. Arrays were then washed and stained with Streptavidin Phycoerythrin in an Affymetrix Fluidics Station 450 according to Affymetrix GeneChip Pico Reagent Kit guide (Thermo Fisher).
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| Scan protocol |
The arrays were scanned using the Affymetrix Gene Chip Scanner 3000 7G and CEL. intensity files were generated by Affymetrix GeneChip Command Console Software (AGCC, Thermo Fisher).
|
| Description |
Gene expression data from microglia of ischemic hemisphere in WT mouse brain at day 2 post 45min MCAo
|
| Data processing |
The Affymetric Clariom S microarrays were analyzed with RMA to detect the gene-level differential expression using the Affymetrix Transcriptome Anaylsis Console (TAC) 4.0 Software using default parameters. The significant genes (FDR<0.05) were ranked by fold-change with a cutoff of 2. TAC4.0 software output does not include data for ~6000 internal control probe sets, thus the sample tables include data for ~22K Clariom S probe sets that assay gene expression.
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| |
|
| Submission date |
Oct 18, 2017 |
| Last update date |
Jul 02, 2019 |
| Contact name |
Katrin I Andreasson |
| E-mail(s) |
kandreas@stanford.edu
|
| Organization name |
Stanford University
|
| Department |
Neurology and neurological sciences
|
| Lab |
kAT
|
| Street address |
1201 Welch Rd, MSLS P250
|
| City |
Stanford |
| State/province |
California |
| ZIP/Postal code |
94305 |
| Country |
USA |
| |
|
| Platform ID |
GPL23038 |
| Series (1) |
| GSE105132 |
Differential gene expression in TREM1 -/- vs +/+ myeloid cells 48h after MCAo-RP |
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