|
| Status |
Public on Jul 11, 2013 |
| Title |
Ctrl OBCs, post doxycyclin withdrawal, rep4 |
| Sample type |
RNA |
| |
|
| Source name |
Control osteoblastic lineage cells 5-6 weeks post doxycylin withdrawal
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Osteoblastic lineage cell
strain: C57BL/6
phenotype: Control
time point: 5-6 weeks post doxycylcin withdrawal
|
| Treatment protocol |
Crushed bones were washed with HBSS until the bone chips were white and endosteal stromal cells were released by digestion for 1 hour at 37°C at 110 rpm with 3 mg/ml type I collagenase (Worthington) dissolved in HBSS. The released endosteal cells were washed with HBSS + 2% FBS, and residual bone material was removed by filtering through a 45 μm filter. Cells were stained with unconjugated rat anti-lineage (B220, CD3, CD4, CD5, CD8, Ter119, Mac-1, Gr-1) antibodies followed by goat anti-rat-Cy5PE or Qdot605 secondary antibodies and directly conjugated CD31-FITC, Sca-1-PB, CD45- Cy7APC and CD51-bio/streptavidin-APC antibodies. Cells were sorted on a FACS ARIAII upon PI exclusion of dead cells. OBCs were single-sorted and directly collected in Trizol.
|
| Growth protocol |
Control and BA mice were withdrawn from doxycycline at 5 week of age to induce MPN development in BA mice. Five to 6 weeks later, bones were harvested for OBC isolation and RNA extraction
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from 500-2300 purified OBCs using the Arcterus PicoPure RNA kit and a modified protocol for Trizol samples according to manufacturers’ instruction. RNA was then amplified using the Nugen Pico WTA kit and amplified products were cleaned using the Qiagen QiaQuick PCR purification kit following Nugen’s protocols.
|
| Label |
Biotin
|
| Label protocol |
Sense-strand cDNA targets were generated using the Nugen WT-Ovation Exon Module, and then fragmented and biotinylated using the Nugen Encore Biotin Module according to manufacturers’ instructions.
|
| |
|
| Hybridization protocol |
Biotinylated cRNA were prepared according to the GeneChip Expression Analysis Technical Manual 702232 Rev. 3
|
| Scan protocol |
GeneChips were scanned using the Affymetric GCS 3000 Scanner
|
| Data processing |
Data was processed using AGCC 3.2.2. Data were GC-RMA normalized and statistically significant differentially expressed genes were determined using Significance Analysis of Microarrays (SAM) (Tusher et al., 2001).
|
| |
|
| Submission date |
Jul 01, 2013 |
| Last update date |
Jul 11, 2013 |
| Contact name |
Emmanuelle Passegué |
| Organization name |
University of California San Francisco
|
| Department |
Medicine
|
| Lab |
Passegue Lab
|
| Street address |
35 Medical Center Way, RMB-1017 Pod B, box 0667
|
| City |
San Francisco |
| State/province |
California |
| ZIP/Postal code |
94143 |
| Country |
USA |
| |
|
| Platform ID |
GPL6246 |
| Series (1) |
| GSE48438 |
Expression data from osteoblastic lineage cells isolated from normal and leukemic mice |
|
