|
Status |
Public on Oct 03, 2008 |
Title |
25 hours NIH3T3_Forskolin_25 |
Sample type |
RNA |
|
|
Source name |
NIH3T3 fibroblasts
|
Organism |
Mus musculus |
Characteristics |
synchronized with forskolin
|
Treatment protocol |
Cells were synchronized with forskolin 20 hours before samples were collected.
|
Growth protocol |
NIH3T3 cells (ATCC) were grown to confluence in DMEM supplimented with 10% FCS and P/S/G on 35mm dishes.
|
Extracted molecule |
total RNA |
Extraction protocol |
Cells were washed briefly with 1X PBS. 1 mL Trizol (Invitrogen) was used to homogenize cells, and samples were frozen at -80 until total RNA was purified.
|
Label |
biotin
|
Label protocol |
Second-strand cDNA synthesis was followed by in vitro transcription for linear amplification of each transcript and incorporation of biotinylated CTP and UTP.
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Hybridization protocol |
The cRNA products were fragmented to 200 nucleotides or less, heated at 99 degrees C for 5 min and hybridized for 16 h at 45 degrees C. The microarrays were then washed at low (6X SSPE) and high (100mM MES, 0.1M NaCl) stringency and stained with streptavidin-phycoerythrin. Fluorescence was amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain.
|
Scan protocol |
A confocal scanner was used to collect fluorescence signal at 3um resolution after excitation at 570 nm. The average signal from two sequential scans was calculated for each microarray feature.
|
Description |
n/a
|
Data processing |
Affymetrix Microarray Suite 5.0 was used to quantitate expression levels for targeted genes; default values provided by Affymetrix were applied to all analysis parameters. Border pixels were removed, and the average intensity of pixels within the 75th percentile was computed for each probe. The average of the lowest 2% of probe intensities occurring in each of 16 microarray sectors was set as background and subtracted from all features in that sector. Probe pairs were scored positive or negative for detection of the targeted sequence by comparing signals from the perfect match and mismatch probe features.
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Submission date |
Jun 27, 2008 |
Last update date |
Aug 28, 2018 |
Contact name |
Michael Hughes |
E-mail(s) |
michael.evan.hughes@gmail.com
|
Organization name |
UPenn
|
Street address |
421 Curie Bvd, Brb 835
|
City |
Philadelphia |
State/province |
PA |
ZIP/Postal code |
19104 |
Country |
USA |
|
|
Platform ID |
GPL1261 |
Series (1) |
GSE11922 |
High temporal resolution profiling of NIH3T3 cells |
|
Relations |
Reanalyzed by |
GSE119085 |