|
| Status |
Public on Nov 13, 2009 |
| Title |
Wild type +SU5402 (5μM) rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Wild type +SU5402 (5μM)
|
| Organism |
Mus musculus |
| Characteristics |
cell line: Wild type ES cells
treatment: SU5402 (5μM) 24 hours
|
| Treatment protocol |
Cells were treated 24h with either heparan sulfation inhibitor (NaClO3-, 20mM) or FGFR inhibitor (SU5402, 5μM)
|
| Growth protocol |
Cells were cultured on gelatin coated plates in Glascow MEM (Sigma-Aldrich) supplemented with 15% FBS (Hyclone), 1000 IU/ml LIF, 2-mercaptoethanol, and Non-Essential Amino Acids (Gibco). Medium was changed daily and cells passaged every second day.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from 3 biological replicates of wild-type and Ndst1/2 - /- ES cells subjected to NaCIO3-, SU5402 or control treatments using the RNeasy Mini kit (Qiagen, Germany) according to manufacturer instructions. RNA quality was assessed on the 2100 Bioanalyser (Agilent, USA) using the RNA 6000 Nano Chip kit (Agilent, USA) for intact 18S and 28S ribosomal peaks without significant degradation (RNA Integrity Number >9) for all samples.
|
| Label |
Biotin
|
| Label protocol |
500ng of total RNA from each sample was reverse transcribed into cDNA and in vitro transcribed into biotin-labelled cRNA using the Illumina TotalPrep RNA Amplification kit (Ambion, USA).
|
| |
|
| Hybridization protocol |
750ng of each cRNA 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.
|
| Description |
Biological replicate 2
|
| Data processing |
The microarray data was subjected to background subtraction 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 wild type ES cell controls with a cutoff at 1.5-fold change. Reference: 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.
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| |
|
| Submission date |
May 06, 2009 |
| Last update date |
Nov 13, 2009 |
| 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) |
| GSE15974 |
Heparan Sulfation Dependent FGF Signalling Maintains ES Cells Primed for Differentiation in a Heterogeneous State |
|
