|
| Status |
Public on Jun 30, 2011 |
| Title |
E14TG2a ES cells +10uM SB-431542 biological rep3 |
| Sample type |
RNA |
| |
|
| Source name |
HPRT- E14TG2a ES cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: E14TG2a ES cells
cell passage: P23
treatment group: +10uM SB-431542
treatment duration: 18 hours treatment
|
| Treatment protocol |
Cells were pretreated 6h with 20% KSR in DMEM containing 100μM non essential amino acids, 100U/ml penicillin, 100μg/ml streptomycin, 2mM GlutaMAX I (Invitrogen), 55μM β mercaptoethanol (Sigma) supplemented with 10uM SB-431542. This was followed by 18h in the same media supplemented with 25ng/ml Activin (ACT), 10uM SB-431542 (SB), 1/5000 DMSO or unsupplemented (KSR).
|
| Growth protocol |
Cells were cultured in 20% ES cell qualified FBS in DMEM containing 100μM non essential amino acids, 100U/ml penicillin, 100μg/ml streptomycin, 2mM GlutaMAX I (Invitrogen), 55μM β mercaptoethanol (Sigma) and 1X homemade Leukemia Inhibitory factor (LIF). Medium was changed daily and cells passaged every second day.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from 4 biological replicates of HPRT- E14TG2a ES cells subjected to ACT, SB or DMSO/KSR control treatments using the RNeasy Mini kit (Qiagen) according to manufacturer instructions. RNA quality was assessed on the 2100 Bioanalyser (Agilent) using the RNA 6000 Nano Chip kit (Agilent) 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).
|
| |
|
| Hybridization protocol |
750ng of each cRNA sample was hybridized to MouseRef-8 v1.1 expression beadchip microarrays (Illumina) as per manufacturer specifications.
|
| Scan protocol |
Microarrays were scanned on the BeadArray Reader (Illumina) at scan factor 1 as per manufacturer specifications.
|
| Description |
Biological replicate 3
|
| Data processing |
The microarray data was subjected to background subtraction on the BeadStudio Data Analysis software (Illumina) and normalized using the Cross-correlation method (Chua et al., 2006) with MATLAB scripts. Differentially expressed transcripts were identified based on the mean Log2 fold-change in expression values compared to the control DMSO treatment 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 |
Jul 28, 2010 |
| Last update date |
Jun 30, 2011 |
| 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 |
GPL6103 |
| Series (1) |
| GSE23239 |
Graded Nodal/Activin Signaling Governs ES Cell Fate Decisions via Differential Recruitment of Phospho-Smad2 to Oct4 and Distinct Target Gene Subsets |
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