|
| Status |
Public on Mar 18, 2008 |
| Title |
CellBody,FN,rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Cell body from fibronectin-stimulated cells
|
| Organism |
Mus musculus |
| Characteristics |
NIH/3T3 fibroblasts
|
| Treatment protocol |
NIH/3T3 cells (~1x106 ) were placed in serum-free media in the upper compartment of a Transwell insert equipped with a 3mm porous polycarbonate membrane whose underside only was coated with 5mg/ml fibronectin. Cells were allowed to extend pseudopodial protrusions towards the fibronectin-coated surface for 1 hr, they were briefly rinsed with PBS and fixed with 0.3 % methanol-free formaldehyde (Polysciences, Inc) in PBS for 10 min at room temperature. Glycine was added to 250 mM for 5 min at room temperature and the cells were washed twice with PBS. To isolate pseudopodia, cell bodies on the upper membrane surface were manually removed with cotton swab and laboratory paper and pseudopodia on the underside of the membrane were scraped into crosslink reversal buffer (100mM Tris pH 6.8, 5mM EDTA, 10mM DTT and 1% SDS). Cell bodies were similarly isolated except that pseudopodia on the underside of the membrane were manually removed and cell bodies were scraped into crosslink reversal buffer. Extracts were incubated at 70oC for 45 min to reverse the formaldehyde-induced crosslinks.
|
| Growth protocol |
NIH/3T3 cells grown in DMEM supplemented with 10% calf serum, sodium pyruvate, penicillin and streptomycin
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Trizol LS reagent (Invitrogen) according to the manufacturer's instructions
|
| Label |
biotin
|
| Label protocol |
Standard Affymetrix protocol (Affymetrix GeneChip Expression Analysis Technical Manual, rev.3. 2001)
|
| |
|
| Hybridization protocol |
Standard affymetrix protocol (Affymetrix GeneChip Expression Analysis Technical Manual, rev.3. 2001)
|
| Scan protocol |
GeneChips were scanned using the Hewlett-Packard GeneArray Scanner
|
| Description |
06-23_CB_B
|
| Data processing |
Data were analyzed using dChip software version 1.2. PM/MM model was used with either median value of 10th percentile of the intensity of Absent genes or 20, whichever larger.
|
| |
|
| Submission date |
Jan 22, 2008 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Stavroula Mili |
| E-mail(s) |
[email protected]
|
| Phone |
434-982-0083
|
| Organization name |
University of Virginia
|
| Department |
Microbiology
|
| Lab |
Dr Ian G Macara
|
| Street address |
1400 Jefferson Park Avenue
|
| City |
Charlottesville |
| State/province |
VA |
| ZIP/Postal code |
22903 |
| Country |
USA |
| |
|
| Platform ID |
GPL1261 |
| Series (2) |
| GSE10227 |
Analysis of RNA distribution between pseudopodia and cell bodies in response to Fibronectin |
| GSE10230 |
Analysis of RNA distribution between pseudopodia and cell bodies in response to LPA stimulation or fibronectin |
|
| Relations |
| Reanalyzed by |
GSE119085 |
