|
| Status |
Public on Oct 26, 2014 |
| Title |
MPP control vector (MSCV) rep2 |
| Sample type |
RNA |
| |
|
| Source name |
WT MPPs
|
| Organism |
Mus musculus |
| Characteristics |
genotype/variation: WT expressing MSCV-GFP
cell type: multipotent progenitors
|
| Treatment protocol |
The RNA samples of GFP+ WT MPPs (Lin-, c-kit+, Sca-1+ Flt3mid) infected with MSCV-GFP or Bach2 GFP retrocirus vectors. cells were pooled from WT mouse (4 mice per one experiment). MPPs were stimulated overnight with SCF (50ng/ml), IL-3(5ng/ml) and IL-6(10ng/ml), and were spin infected on the nextday. Infected cells were cultured under the Multi-lineage developmental condition 2d.Then sorted by GFP+ expression.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the RNeasy Plus Micro kit (QIAGEN) following the manufacturer's recommendations. RNA quantity and quality were measured with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Three RNA samples of CLPs or MPPs were performed in two rounds of amplification using the Amino Allyl MessageAmpII aRNA Amplification Kit (Ambion) and Cy3 labeled with a CyDye post-labeling Reactive Dye Pack (Amersham) following the manufacturer’s protocol. After amplification and labeling, cRNA yields and dye incorporation efficiencies were determined using a Nanodrop spectrophotometer.
|
| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity > 104-156 pmo Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Sureprint G3 Mouse GE 8x60k Microarray slides 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 |
Labeled samples were incubated with Sureprint G3 Mouse GE 8x60k Microarray Kit slides for 17 h. The slides were scanned, and the gene expression analysis was performed using the Genespring GX software program version 11.
|
| Description |
Gene expression in MPPs expressing MSCV-GFP2
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Sep 25, 2014 |
| Last update date |
Oct 26, 2014 |
| Contact name |
ari itoh |
| Organization name |
Tohoku univ.
|
| Street address |
2-1 Seiryo-machi, Aobaku
|
| City |
Sendai |
| State/province |
Miyagi |
| ZIP/Postal code |
980-8575 |
| Country |
Japan |
| |
|
| Platform ID |
GPL13912 |
| Series (1) |
| GSE61409 |
The transcription repressors Bach2 and Bach1 promote B cell development by repressing the myeloid program |
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