|
| Status |
Public on Mar 07, 2018 |
| Title |
zeb1-kd-microglia-after mcao-rep2 |
| Sample type |
RNA |
| |
|
| Source name |
zeb1-kd-microglia-after mcao
|
| Organism |
Mus musculus |
| Characteristics |
tissue: microglia
gender: male
age: 8 weeks
genotype: ZEB1-knock down
treatment: tMCAO
|
| Treatment protocol |
Microglia cells from the brains of wild type mice, ZEB1-overexpression and ZEB1-knock down mice were isolated by MACS sorting using a mouse CX3CR1 cell enrichment kit before and after tMCAO.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the Trizol reagent (Invitrogen) following the manufacturer's recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
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| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression of zeb1-kd micorglia after tMCAO
MCAO-06
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
Feb 05, 2018 |
| Last update date |
Mar 07, 2018 |
| Contact name |
Junwei Hao |
| E-mail(s) |
hjw@tmu.edu.cn
|
| Organization name |
Tianjin Medical University General Hospital
|
| Street address |
154 Anshan Road, Heping District
|
| City |
Tianjin |
| ZIP/Postal code |
300052 |
| Country |
China |
| |
|
| Platform ID |
GPL21163 |
| Series (1) |
| GSE110141 |
Gene expression signatures before or after transient middle cerebral artery oclussion (tMCAO) in ZEB1 transgenic and wild type mice |
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