|
| Status |
Public on Mar 08, 2011 |
| Title |
Spliceostatin treatment replicate 2 Dye-Swap |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
HeLa spliceostatin treatment replicate 2
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
agent: spliceostatin
|
| Treatment protocol |
Before treatment, media was removed, cell were washed twice with 1 ml 1X PBS, added 1 ml DMEM without serum and antibiotics and treat the cells with spliceostatin 100 ng/ml for three hours.
|
| Growth protocol |
Cells were grown in a 6-well plate with DMEM glutamax (Invitrogen 61965-059) in the presence of 10% serum and penicillin/streptomycin until 80% confluence.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNeasy mini kit (QUIAGEN, cat.no. 74104) and DNase treated on-column with DNase RNase-free
Quality assesment of Total RNA samples were analyzed using the Agilent Bioanalyzer 2100 and the RNA 6000 LabChip Kit (Agilent) with the Eukaryote Total RNA Nano Assay.
|
| Label |
Cy3
|
| Label protocol |
Sample was labeled using the Quick-Amp labeling kit, no dye (Agilent 5190-0447) and cyanine 5-CTP and cyanine 3-CTP (Perkin-Elmer NEL580, NEL581). Purified using Quiagen RNeasy mini kit. Quality control assessed by Agilent Bioanalyzer 2100.
|
| |
|
| Channel 2 |
| Source name |
HeLa methanol treatment replicate 2
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
agent: methanol
|
| Treatment protocol |
Before treatment, media was removed, cell were washed twice with 1 ml 1X PBS, added 1 ml DMEM without serum and antibiotics and treat the cells with methanol for three hours.
|
| Growth protocol |
Cells were grown in a 6-well plate with DMEM glutamax (Invitrogen 61965-059) in the presence of 10% serum and penicillin/streptomycin until 80% confluence.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNeasy mini kit (QUIAGEN, cat.no. 74104)
Quality assesment of Total RNA samples were analyzed using the Agilent Bioanalyzer 2100 and the RNA 6000 LabChip Kit (Agilent) with the Eukaryote Total RNA Nano Assay.
|
| Label |
Cy5
|
| Label protocol |
Sample was labeled using the Quick-Amp labeling kit, no dye (Agilent 5190-0447) and cyanine 5-CTP and cyanine 3-CTP (Perkin-Elmer NEL580, NEL581). Purified using Quiagen RNeasy mini kit. Quality control assessed by Agilent Bioanalyzer 2100.
|
| |
|
| |
| Hybridization protocol |
Slides were hybridized following manufacturer protocol, with the exception that we hybridized 6 micrograms of each amplified cRNA sample and the Agilent hybridization oven was set to 60ºC.
|
| Scan protocol |
Fluorescent images were obtained using the G2565BA Microarray Scanner System (Agilent) with 100% laser power and 100% PMT settings and 16-bit TIFF images, one for each channel, were quantified using GenePix Pro 6.0 microarray analysis software (Molecular Devices, www.moleculardevices.com).
|
| Description |
Mean foreground and background intensities were extracted from the red (Cy5) and green (Cy3) channels for every spot on the microarray. The background intensities are used to correct the foreground intensities for local variation on the array surface, resulting in corrected red and green intensities.
|
| Data processing |
Raw data were processed using SAPO (http://bioinfo.ciberehd.org/SAP.ang.htm) and CGEM (http://bioinfo.ciberehd.org/CGEM.ang.html) alternative splicing analysis tools.
|
| |
|
| Submission date |
Jun 29, 2010 |
| Last update date |
Mar 09, 2011 |
| Contact name |
BELEN MINANA |
| E-mail(s) |
[email protected]
|
| Phone |
0034933160276
|
| Organization name |
Center for genomic regulation
|
| Department |
GENE REGULATION, STEM CELLS AND CANCER
|
| Lab |
REGULATION OF ALTERNATIVE SPLICING
|
| Street address |
dr aiguader 88
|
| City |
barcelona |
| State/province |
barcelona |
| ZIP/Postal code |
08003 |
| Country |
Spain |
| |
|
| Platform ID |
GPL10635 |
| Series (1) |
| GSE22614 |
Spliceostatin treatment on HeLa cells |
|
