|
| Status |
Public on Mar 13, 2014 |
| Title |
Clone#3_IgG_rep5 |
| Sample type |
RNA |
| |
|
| Source name |
ERK1-GFP human keratinocytes_clone#3_IgG
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: hTERT-immortalized normal human keratinocytes
tranfected with: ERK1-green fluorescent protein chimera
clone id: clone#3
treated with: nonspecific mouse IgG (control)
|
| Treatment protocol |
Treatments were performed in KSF supplemented with bovine pituitary extract (no epidermal growth factor). Conditions included 10ng/mL of TGFα used for ligand-activation; 10ug/mL of monoclonal antibody EGF antagonist mAB225 was used to shut down autocrine-activated ERK1; and nonspecific mouse IgG was used as a control for the autocrine-activated ERK1. Cells were harvested for RNA extraction after 24hrs. Three replicates were collected.
|
| Growth protocol |
hTERT-immortalized normal human keratinocytes (provided by Dr. Jerry Shay, The University of Texas Southwestern Medical Center); Maintained in Keratinocyte serum-free medium (KSF) supplemented with bovine pituitary extract and epidermal growth factor. Stable RNA transfection of ERK1-green fluorescent protein chimera was achieved using Phoenix retroviral packing cell system and puromycin selection. Stable subclones were isolated with distinct ERK activation/oscillation patterns: Clone #1 exhibits transient ERK activation with ligand activation but does not oscillate; Clone #2 exhibits persistent ERK oscillations that are dependent on ligand activation; and Clong #3 exhibits spontaneous ERK oscillations in the absence of ligand activation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was collected using the RNeasy Kit (Qiagen, Valencia, CA).
|
| Label |
biotin
|
| Label protocol |
Complementary DNA was synthesized from 1 µg of total RNA in the presence of an oligo-dT primer containing a T7 RNA polymerase promoter, and an in vitro transcription reaction was performed in the presence of a mixture of biotin-labeled ribonucleotides to produce biotinylated cRNA from the cDNA template, according to manufacturer’s protocols (Affymetrix One-Cycle Target Labeling Kit).
|
| |
|
| Hybridization protocol |
Biotin-labeled cRNA (12 µg) was fragmented to a size range between 50-200 bases for array hybridization. After hybridization, the arrays were washed and stained with streptavidin-phycoerythrin.
|
| Scan protocol |
The arrays were scanned at a resolution of 2.5 microns using an Affymetrix GeneChip Scanner 3000.
|
| Description |
TW45
Clone #3 with autocrine activation
|
| Data processing |
Raw intensity data were quantile normalized using Genespring software by Robust Multi-Array Analysis (RMA) summarization and probes were subject to quality control to measure the efficiency of transcription, integrity of hybridization, and consistency of qualitative calls.
|
| |
|
| Submission date |
Mar 12, 2014 |
| Last update date |
Mar 13, 2014 |
| Contact name |
Katrina M Waters |
| E-mail(s) |
katrina.waters@pnl.gov
|
| Organization name |
Pacific Northwest National Laboratory
|
| Department |
Biological Sciences Division
|
| Street address |
902 Battelle Blvd; MSIN J4-18
|
| City |
Richland |
| State/province |
WA |
| ZIP/Postal code |
99352 |
| Country |
USA |
| |
|
| Platform ID |
GPL571 |
| Series (1) |
| GSE55823 |
ERK Oscillation-Dependent Gene Expression Patterns and Deregulation By The Stress-Response |
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