|
| Status |
Public on Jul 18, 2011 |
| Title |
6h EGF replicate 3 (Illumina) |
| Sample type |
RNA |
| |
|
| Source name |
Cervical cancer cell line (HeLa)
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
treatment: serum deprived for 24 hours, followed by exposure to EGF for 6 hours
|
| Treatment protocol |
For treatments, the cells were transferred to 60 mm dishes and cultured for 48h in complete growth medium, after which they were starved for 24h in DMEM containing 2% FBS. After serum deprivation cells were stimulated with EGF (150 ng/ml) for the indicated times. Three biological replicate experiments were performed, each comparing EGF treated with untreated control HeLa cells.
|
| Growth protocol |
HeLa cells were cultured at 37ºC in a 95/5 Air/CO2 water saturated atmosphere in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat inactivated foetal bovine serum (FBS), 2 mM L-glutamine and 100U/ml Penicilin/streptomycine (complete growth medium).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from HeLa cells was extracted using the RNeasy RNA Isolation kit (Qiagen) followed by treatment with RNase-free DNase I (Ambion, Austin, TX) following manufacturer's instructions. RNA quantification was performed with a Nanodrop spectrophotometer, and the RNA integrity was assessed using the Bioanalyzer 2100 nanoelectrophoresis Lab-on-a–chip system (Agilent, Wilmington, DE). All RNAs used had RIN (RNA integrity number) ranging between 9.3 and 10, and 28S/18S ratios between 1.67 and 2.07.
|
| Label |
biotin / streptavidin-Cy3
|
| Label protocol |
For each sample 200 ng of total RNA were reverse transcribed, amplified by in vitro transcription and labelled with biotin-UTP using the Illumina Total Prep RNA amplification kit (IL1791, Ambion) following the manufacturer's instructions. Labelled sample quality was assessed by spectrophotometry and bioanalyzer. After preheating at 65ºC for 5 min.
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| |
|
| Hybridization protocol |
For each sample, 750 ng of biotinylated cRNA were hybridized in a BeadChip Hyb Chamber with rocking 16 h at 58ºC. The day after the bead arrays were washed with Illumina proprietary washing solutions in a Hybex waterbath: first with static incubation for 10 min at 55ºC in E1BC solution, followed by 10 rinses by dipping in the same solution and shaking 5 min at 90 rpm in an orbital shaker; the next wash was by dipping 10 times in 100% ethanol and shaking 10 min at 110 rpm in an orbital shaker; this was followed by another wash in E1BC solution with 10 dippings followed by 2 min shaking at 90 rpm. Washed bead arrays were blocked in E1 buffer 10 min in rocking incubator and 10 additional minutes with 2 ml of E1 buffer plus streptavidin-Cy3. The fluorescent reagent was washed away with E1BC solution with 10 dippings plus 5 min shaking at 140 rpm. Finally beadarrays were dried by centrifugation 4 min at 275 rcf, followed by scanning in a Illumina Beadstation.
|
| Scan protocol |
The Beadscan software was used to control de scanner, generate .tif images and extract the raw data as tabulated text files. Default settings for DirectHyb Gene Expression were used.
|
| Description |
HeLa cells, serum deprived for 24 hours, followed by exposure to EGF for 6 hours
6h time point, EGF, biological replicate 3 of 3
|
| Data processing |
The raw data was summarized per probe using BeadStudio software Gene Expression module and the summary data file was processed using the PILLA web einterface tool (Lozano et al, unpublished), an implementation of the Lumi package (Du et al, 2008) developed within the Bioconductor project in the R statistical programming environment (Gentleman et al, 2004). Data were normalized using the rsn method and vst as the variance stabilization method. The log2 intensities were median centered and log ratios were computed as differences in log2 intensities for each probe. The SAM (significance analysis of microarrays) two class unpaired comparison test was applied with 100 permutations to detect statistically significant differences in gene expression between treated and control conditions (Tusher et al, 2001).
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| |
|
| Submission date |
Jul 29, 2009 |
| Last update date |
Jul 18, 2011 |
| Contact name |
Lauro Sumoy |
| E-mail(s) |
lsumoy@igtp.cat
|
| Organization name |
IGTP
|
| Department |
High Content Genomics and Bioinformatics
|
| Street address |
Ctra. Can Ruti, Camí de les escoles s/n
|
| City |
Badalona |
| State/province |
Barcelona |
| ZIP/Postal code |
08916 |
| Country |
Spain |
| |
|
| Platform ID |
GPL6102 |
| Series (1) |
| GSE17403 |
Multiple platform assessment of the EGF dependent transcriptome by microarray and deep tag sequencing analysis |
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