|
| Status |
Public on Mar 18, 2008 |
| Title |
CellBody,LPA,rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Cell body from LPA-stimulated cells
|
| Organism |
Mus musculus |
| Characteristics |
NIH/3T3 fibroblasts
|
| Treatment protocol |
NIH/3T3 cells were serum-starved overnight and 1.5x106 cells were placed in the upper compartment of a Transwell insert (24mm diameter, Costar) equipped with a 3mm porous polycarbonate membrane coated on both sides with 5mg/ml fibronectin. Cells were allowed to spread on the upper surface of the membrane for 2 hrs. LPA (150ng/ml) was then added in the bottom chamber to induce the cells to extend pseudopodial protrusions. After 1 hr the cells 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 |
05-61_CB_A
|
| Data processing |
The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method.
|
| |
|
| 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) |
| GSE10226 |
Analysis of RNA distribution between pseudopodia and cell bodies in response to LPA stimulation |
| GSE10230 |
Analysis of RNA distribution between pseudopodia and cell bodies in response to LPA stimulation or fibronectin |
|
| Relations |
| Reanalyzed by |
GSE119085 |
