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Sample GSM877121 Query DataSets for GSM877121
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

Data table header descriptions
ID_REF
VALUE Background subtracted cross-correlation method normalised intensity values generated using MATLAB scripts
Hypoxia 2 Detection Pval

Data table
ID_REF VALUE Hypoxia 2 Detection Pval
ILMN_2896528 4122.0838 0
ILMN_2721178 773.63762 0
ILMN_3033922 1120.9039 0
ILMN_3092673 3892.474 0
ILMN_2816356 53.32554 0
ILMN_2808939 765.52996 0
ILMN_2634564 489.57566 0
ILMN_2737647 20 0.4874687
ILMN_2734484 1053.1585 0
ILMN_2952292 61.775362 0
ILMN_2699078 20 0.3270677
ILMN_1213681 400.62608 0
ILMN_2735413 20 0.1265664
ILMN_2735415 20 0.6240602
ILMN_2891688 651.01577 0
ILMN_2637698 1523.0949 0
ILMN_2674228 827.80779 0
ILMN_2601546 20 0.05513784
ILMN_1230831 20 0.9310777
ILMN_2848071 20 0.1942356

Total number of rows: 25697

Table truncated, full table size 662 Kbytes.




Supplementary data files not provided
Processed data included within Sample table
Raw data are available on Series record

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