|
| Status |
Public on Jul 11, 2008 |
| Title |
mESCs_p400KD_rep4 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
p400 KD mouse embryonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
Mouse embryonic stem cell line E14
|
| Treatment protocol |
One well of a 6-well dish of E14 cells was treated with purified esiRNA (0.33 ng/ul) and Lipofectamine 2000 (Invitrogen) for 72 hr to knockdown expression of p400.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using Trizol reagent combined with PureLink Micro-to-Midi columns (Invitrogen).
|
| Label |
Cy3
|
| Label protocol |
Total RNA quality was assessed using a Pico Chip on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). RNA was amplified and labeled with Cy3-CTP and Cy5-CTP using the Agilent low RNA input fluorescent linear amplification kits following the manufacturers protocol (Agilent).
|
| |
|
| Channel 2 |
| Source name |
EGFP control KD mouse embryonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
Mouse embryonic stem cell line E14
|
| Treatment protocol |
One well of a 6-well dish of E14 cells was treated with purified, control (EGFP) esiRNA (0.33 ng/ul) and Lipofectamine 2000 (Invitrogen) for 72 hr.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using Trizol reagent combined with PureLink Micro-to-Midi columns (Invitrogen).
|
| Label |
Cy5
|
| Label protocol |
Total RNA quality was assessed using a Pico Chip on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). RNA was amplified and labeled with Cy3-CTP and Cy5-CTP using the Agilent low RNA input fluorescent linear amplification kits following the manufacturers protocol (Agilent).
|
| |
|
| |
| Hybridization protocol |
Labeled cRNA was assessed using the Nandrop ND-100 (Nanodrop Technologies, Inc., Wilmington DE), and equal amounts of Cy3 and Cy5 labeled target were hybridized to Agilent whole mouse genome 4x44K Ink-jet arrays (Agilent). Hybridization samples were paired according to group designation and dye swaps were incorporated to correct for any dye bias. Hybridizations were performed for 14 hrs, according to the manufacturers protocol (Agilent).
|
| Scan protocol |
Arrays were scanned using the Agilent microarray scanner (Agilent) and raw signal intensities were extracted with Feature Extraction v8.9 software (Agilent).
|
| Description |
Sample preparation, labeling, and array hybridizations were performed according to standard protocols from the UCSF Shared Microarray Core Facilities and Agilent Technologies (http://www.arrays.ucsf.edu and http://www.agilent.com).
|
| Data processing |
This dataset was normalized using robust locally weighted regression (loess) to correct for intensity, spatial and other dye biases (Cleveland 1979, JASA 74(36): 829; Yang et al 2002, NAR 30(4)). This procedure was performed and carried out using the Bioconductor package “marray” (http://www.bioconductor.org). In the context of microarray experiments, loess normalization captures the non-linear dependence of the intensity log-ratio M = log2R-log2G = log2(R/G) on the overall intensity A= (log2R+log2G)/2.
|
| |
|
| Submission date |
Apr 22, 2008 |
| Last update date |
Jun 30, 2008 |
| Contact name |
Jason T Huff |
| Organization name |
University of California, Berkeley
|
| Department |
Plant & Microbial Biology
|
| Lab |
Daniel Zilberman
|
| Street address |
211 Koshland Hall
|
| City |
Berkeley |
| State/province |
CA |
| ZIP/Postal code |
94720 |
| Country |
USA |
| |
|
| Platform ID |
GPL4134 |
| Series (2) |
| GSE11240 |
An RNAi Screen of Chromatin Proteins Identifies Tip60-p400 as a Regulator of Embryonic Stem Cell Identity, Experiment A |
| GSE11243 |
An RNAi Screen of Chromatin Proteins Identifies Tip60-p400 as a Regulator of Embryonic Stem Cell Identity |
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