|
| Status |
Public on Dec 31, 2009 |
| Title |
ES Cells+N2B27 medium+Dox 6 hrs 3b |
| Sample type |
RNA |
| |
|
| Source name |
ES cells, N2B27 medium+Dox, 6 hrs
|
| Organism |
Mus musculus |
| Characteristics |
treatment: Dox
gender: Male
strain: Ainv15
cell type: ES cells
developmental stage: Blastocyst
|
| Treatment protocol |
2,000,000 cells were plated into gelatin-coated 10cm dishes in ESC medium or N2B27 medium consisting of a 1:1 ratio of DMEM/F12 and Neurobasal media supplemented with 0.5% N2 (Gibco) 0.5% B27 (GIBCO, San Diego, California, United States), and 2-mercaptoethanol and glutamine. For the relevant treatments doxycycline was used at a concentration of 2ug/ml (SIGMA) and DAPT at 2.5ng/ml (Calbiochem). In experiments where Notch signaling was pharmacologically blocked, DAPT in ESC medium was applied 4 hours before initiation of array conditions. Hypoxic culture was achieved with an Invivo2 Hypoxia Workstation 400 at 1% oxygen pressure, 6% CO2 37˚C.
|
| Growth protocol |
ES cells were maintained on gelatin (Sigma) coated dishes (Corning) in ESC medium consisting of DMEM (Gibco) supplemented with 5% ESC tested FCS (Sigma), 5% KSR (Gibco), pyruvate (Gibco), non-essential amino acids, beta-mercaptoethanol and LIF at 6% CO2 at 37˚C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Following the respective treatments, cells were washed with PBS, trypsinised, resuspended in RNAlater and stored at -20°C. Total RNA extraction was performed using the RNeasy Mini kit (Qiagen) according to the 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 labelled cRNA sample was hybridized to MouseRef-8 v1.1 Expression BeadChip microarrays (Illumina) according to manufacturer specifications.
|
| Scan protocol |
Microarrays were scanned on the BeadArray Reader (Illumina, USA) at scan factor 1 as per manufacturer specifications.
|
| Description |
Technical replicate of 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).
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 |
Nov 18, 2009 |
| Last update date |
Dec 22, 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 |
GPL6103 |
| Series (1) |
| GSE19074 |
Integration between Notch- and hypoxia-induced transcriptomes |
|
