|
| Status |
Public on Mar 19, 2010 |
| Title |
wibr2_20A-wibr2_5 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
c29_20% (test)
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: c29
oxygen concentration: 20%A
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from undifferentiated hES colonies between passages 30-40 and isolated using Trizol reagent (Gibco, Invitrogen, Carlsbad, CA) according to the manufacturers protocol. RNA was subsequently treated with DNAse I in order to remove potential genomic DNA contamination using an RNAse free DNAse kit (Zymo Research, Orange County, CA).
|
| Label |
Cy3
|
| Label protocol |
Cy-dye labeled cRNA samples were prepared using Agilent’s QuickAmp sample labeling kit. Input was 2 ug total RNA
|
| |
|
| Channel 2 |
| Source name |
c29_5% rep 2 (ref)
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: c29
oxygen concentration: 5%
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from undifferentiated hES colonies between passages 30-40 and isolated using Trizol reagent (Gibco, Invitrogen, Carlsbad, CA) according to the manufacturers protocol. RNA was subsequently treated with DNAse I in order to remove potential genomic DNA contamination using an RNAse free DNAse kit (Zymo Research, Orange County, CA).
|
| Label |
Cy5
|
| Label protocol |
Cy-dye labeled cRNA samples were prepared using Agilent’s QuickAmp sample labeling kit. Input was 2 ug total RNA
|
| |
|
| |
| Hybridization protocol |
Agilent (human 4x44k) expression arrays were hybridized according to our laboratory method, which differs slightly from the Agilent standard hybridization protocol. The hybridization cocktail consisted of 1.65 ug cy-dye labeled cRNA for each sample, Agilent hybridization blocking components, and fragmentation buffer. The hybridization cocktails were fragmented at 60°C for 30 minutes, and then Agilent 2X hybridization buffer was added to the cocktail prior to application to the array. The arrays were then hybridized for 16 hours at 60°C in an Agilent rotor oven set to maximum speed. The arrays were treated with Wash Buffer #1 (6X SSPE / 0.005% nlaurylsarcosine) on a shaking platform at room temperature for 2 minutes, and then Wash Buffer #2 (0.06X SSPE) for 2 minutes at room temperature. The arrays were then dipped briefly in acetonitrile before a final 30 second wash in Agilent Wash 3 Stabilization and Drying Solution, in the hood using a stir plate and stir bar at room temperature.
|
| Scan protocol |
Arrays were scanned using an Agilent scanner and the data was extracted using Agilent’s Feature Extraction software.
|
| Description |
c29 incubated in 5% oxygen or 20% oxygen acutely
|
| Data processing |
Microarray data were processed, normalized within-array by LOESS and normalized between arrays by Aquantile using Limma package.
Dye swap was done in this experiment so the test samples are sometimes Cy5 or Cy3. 20% and 20%A are test while all 5% are reference.
|
| |
|
| Submission date |
Mar 17, 2010 |
| Last update date |
Mar 18, 2010 |
| Contact name |
Albert W Cheng |
| E-mail(s) |
awcheng@mit.edu
|
| Organization name |
Whitehead Institute
|
| Street address |
Room 453, 9 Cambridge Center
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE20937 |
Derivation of pre-X inactivation human embryonic stem cells under physiological oxygen concentrations |
|
