|
| Status |
Public on Jul 30, 2014 |
| Title |
WT2 Hypoxia 24h 4X10^5 cells/6cm plate |
| Sample type |
RNA |
| |
|
| Source name |
TT2 Mouse ES cells WT2 Hypoxia 24h 4X10^5 cells/6cm plate
|
| Organism |
Mus musculus |
| Characteristics |
gender: Male
strain: TT2 (c57BL/6xCBA)
cell type: ES cells
genotype: Wild-type
treatment: Hypoxia
time: 24h
cell density: 4X10^5 cells/6cm plate
|
| Treatment protocol |
Cells were cultured under hypoxic conditions (1% O2, 5% CO2 at 37°C) in an Invivo2 Hypoxia Workstation 400 hypoxic chamber (Ruskinn Technology) for 4 or 24 hours. Control cells were cultured in normoxic conditions (21% O2, 5% CO2 at 37°C) in a Forma Steri-Cycle CO2 incubator (Thermo Scientific)
|
| Growth protocol |
Mouse ES cells were maintained and expanded in DMEM supplemented with 10% fetal calf serum, LIF (500U/ml), 55uM 2-mercaptoethanol, 2mM GlutaMAX, 0.1mM non-essential amino acids, 100U/ml Penicillin and 100ug/ml Steptomycin (Invitrogen).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from wild-type, JMJD1a KO, G9a KO or reconstituted G9a KO ES cells subjected to 24 hours normoxia, 4 hours hypoxia or 24 hours hypoxia 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 >8) for all samples.
|
| Label |
Cy3
|
| 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 v2.0 Expression BeadChip microarrays (Illumina) 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 on the BeadStudio Data Analysis Software (Illumina) and normalized using the cross-correlation method (Chua et al., 2006). Differential gene expression was identified based on a fold change cutoff of >1.5 compared to the average of the wild-type normoxic controls.
Normalized values represent linear relative data that have not been log transformed. They were obtained from the original microarray raw data after background subtraction on the Illumina Beadstudio platform followed by normalization using the cross-correlation method (Chua et al., 2006) performed on MATLAB.
references: 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 |
Jan 12, 2012 |
| Last update date |
Jul 30, 2014 |
| Contact name |
Kian Leong LEE |
| E-mail(s) |
[email protected]
|
| 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) |
| GSE35061 |
Role of the hypoxia-inducible histone H3K9 methylation regulating enzymes Jmjd1a and G9a in stem cell self-renewal and tumorigenesis |
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