|
| Status |
Public on Nov 29, 2017 |
| Title |
Control_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
P19 cells
|
| Organism |
Mus musculus |
| Characteristics |
cell line: P19
cell type: murine pluripotent stem cells
treated with: none (untreated control)
|
| Treatment protocol |
Cells were treated with 5 nM myxothiazol for 12 h
|
| Growth protocol |
Cells were grown in high glucose DMEM medium (with sodium pyruvate with GlutaMAX, with Phenol Red) supplemented with 10% FBS at 37 ºC in a humidified incubator with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared from 5*10^6 cells using the RNeasy Mini Kit (Qiagen) and RNase-Free DNase Kit (Qiagen) following the manufacturer's recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 150 ng RNA using the one-color Low Input Quick Amp Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (Qiagen). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
0.6 ug of Cy3-labelled cRNA (specific activity >14.0 pmol Cy3/ug cRNA) was fragmented and hybridized using Gene Expression Hybridization Kit following the manufacturer's recommendations for 17 hours at 65°C, in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent); wash buffers contained 0.005% Triton X-102
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent microarray confocal laser scanner (G2505B) using one color scan setting for 8x60K array slides (Scan Area 61 x 21.6 mm, Scan resolution 5µm, Dye channel is set to Green and Green PMT is set to 100%).
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid:074809_D_F_20150624) to obtain background subtracted and spatially detrended Processed Signal intensities.
|
| |
|
| Submission date |
Nov 28, 2017 |
| Last update date |
Jan 23, 2018 |
| Contact name |
Natalia Pashkovskaia |
| E-mail(s) |
pashkovskaia.natalia@gmail.com
|
| Organization name |
Technische Universität Dresden
|
| Street address |
Zellescher Weg 20B
|
| City |
Dresden |
| ZIP/Postal code |
01217 |
| Country |
Germany |
| |
|
| Platform ID |
GPL21163 |
| Series (1) |
| GSE107414 |
Gene expression analysis of murine pluripotent stem cells treated with myxothiazol for 12 h |
|
