|
| Status |
Public on Jul 19, 2010 |
| Title |
6755 |
| Sample type |
RNA |
| |
|
| Source name |
HTO_NIPP1m_rep4
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa-Tet-Off (Clontech) stably transfected with Flag-NIPP1m (point mutant with a disrupted PP1-docking motif, m n°1)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
HTO_parental was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS and 250 µg/ml geneticin, HTO_NIPP1wt and HTO_NIPP1m were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS and 250 µg/ml geneticin, 200 µg/ml hygromycin and 50 ng/ml doxycyclin. HTO_NIPP1wt and HTO_NIPP1m were cultured for 72 hours without doxycycline after which total RNA was isolated from all three cell lines using the Genelute Mammalian Total RNA Miniprep kit.
|
| Label |
Cy3
|
| Label protocol |
1µg of sample was spiked with a set of 10 positive control transcripts (Agilent Technologies), converted to double stranded cDNA in a reverse transcription reaction, amplified and labeled with Cy3 in an in vitro transcription reaction according to the manufacturer's protocol (Agilent Technologies).
|
| |
|
| Hybridization protocol |
28 pmol Cy3 incorporated dye was fragmented and resuspended in hybridization buffer HI-RPM (Agilent Technologies). The arrays were hybridized in microarray hybridization chambers (Agilent Technologies) overnight at 65ºC, rpm=10 for 17 hours.
|
| Scan protocol |
After washing the slides were scanned with a DNA microarray scanner (Agilent Technologies) and images were processed with the Feature Extraction Software (Agilent Technologies).
|
| Description |
Cells: HeLa-Tet-Off (Clontech) stabily transfected with Flag-NIPP1m (point mutant with a disrupted PP1-docking motif, cell line m n°1)
|
| Data processing |
We use the Agilent processed signal values (i.e., feature gProcessedSignal) from Agilent Feature Extraction software and compute log2-intensities. We remove Agilent control probes, as well as probes with signals below background on all arrays (absent spots). To decide whether a signal is significantly above background, we use the feature gIsPosAndSignif, also provided by the Agilent software. In case of multiple probes for the same Agilent ID, log2-intensities are averaged.
|
| |
|
| Submission date |
Dec 24, 2009 |
| Last update date |
Apr 03, 2015 |
| Contact name |
Rekin's Janky |
| E-mail(s) |
[email protected]
|
| Organization name |
VIB
|
| Department |
Nucleomics Core
|
| Street address |
Herestraat 49 Box 816
|
| City |
Leuven |
| ZIP/Postal code |
B-3000 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL6480 |
| Series (2) |
| GSE19642 |
Expression profiling of HeLa Tet-off cells expressing Flag-tagged version of WT NIPP1 or PP1 binding mutant of NIPP1. |
| GSE67558 |
Protein phosphatase PP1-NIPP1 activates mesenchymal genes in HeLa cells |
|
