|
| Status |
Public on Feb 08, 2013 |
| Title |
CD31 Positive Cells + Hypoxia (1.5-2% O2) Replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
CD31 Positive Wild Type Mouse CCE ES Cells+Hypoxia (1.5-2% O2) Replicate 2
|
| Organism |
Mus musculus |
| Characteristics |
gender: Male
cell line: CCE (129/Sv)
cell type: ES cells
genotype: Wild type
marker: CD31 positive
treatment: Hypoxia (1.5-2% O2)
time: 4 days
|
| Treatment protocol |
10,000 ES cells/ml were aggregated as embryoid bodies (EB) in 20 ul drops and differentiated for 4 days in IMDM medium, 15% FBS (Gibco), 150 uM monothioglycerol, and 50 ug/ml Ascorbic acid (Sigma). On day 4, EBs were harvested and dissociated, stained for Flk1 and sorted to purity using flow cytometry. 50,000 Flk1+ cells/ml were plated on collagen type IV (BD Bioscience) coated plates in biological triplicates and differentiated for 4 additional days in normoxia (21% O2) or hypoxia (1.5-2% O2) with or without 4 umol/l L-685.458 (BachemAG) in the same IMDM medium.
|
| Growth protocol |
Wild type mouse CCE ES cells were cultured on gelatin coated plates in Glasgow MEM (Sigma) supplemented with 15% FBS (Hyclone), 1000 IU/ml LIF, 0.1 mM 2-mercaptoethanol and 0.1 mM Non-Essential Amino Acids (Gibco). Medium was changed daily and cells passaged every second day.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
>50,000 CD31+ cells were FACS sorted from each sample and total RNA was extracted using the RNeasy Micro kit (Qiagen, Germany).
|
| Label |
Cy3
|
| Label protocol |
300ng of total RNA from each sample was reverse transcribed into cDNA and in vitro transcribed into biotin-labeled cRNA using the Illumina TotalPrep RNA Amplification kit (Ambion, USA).
|
| |
|
| Hybridization protocol |
750ng of cRNA from each sample was hybridized to MouseRef-8 v2.0 Expression BeadChip microarrays (Illumina, USA) as per manufacturer specifications.
|
| Scan protocol |
Microarrays were scanned on the BeadArray Reader (Illumina, USA) at scan factor 1 as per manufacturer specifications.
|
| Data processing |
Raw intensity values were subjected to background subtraction performed on the BeadStudio Data Analysis software (Illumina, USA) and normalized using the Cross-correlation method (Chua et al., 2006). Differentially expressed transcripts were identified based on the mean Log2 fold change in expression values compared to the average of the normoxia controls, with a minimum cutoff at 1.5-fold change.
Chua SW, Vijayakumar P, Nissom PM, Yam CY, Wong VV, Yang H: A novel normalization method for effective removal of systematic variation in microarray data. Nucleic acids research 2006, 34(5):e38.
|
| |
|
| Submission date |
Feb 16, 2012 |
| Last update date |
Feb 08, 2013 |
| Contact name |
Kian Leong LEE |
| E-mail(s) |
kianleong.lee@duke-nus.edu.sg
|
| Phone |
+(65) 6601 3685
|
| Organization name |
National University of Singapore (NUS)
|
| Department |
Duke-NUS Medical School
|
| Lab |
Cancer & Stem Cell Biology Program (CSCB)
|
| Street address |
#07-21, 8 College Road
|
| City |
Singapore |
| State/province |
Singapore |
| ZIP/Postal code |
169857 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL6885 |
| Series (1) |
| GSE35894 |
Hypoxia-induced Arterial Differentiation Requires Adrenomedullin and Notch Signaling |
|
