|
| Status |
Public on Aug 27, 2013 |
| Title |
Compound 3 treated 48h rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
MDA-MB-231 treated with EZH2 inhibitor compound 3 for 48h
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MDA-MB-231
|
| Treatment protocol |
Cell cultures were treated for either 48h or 72h with an EZH2/G9a inhibitor compound. Compounds were diluted in DMSO at Stocks of 10mM.
|
| Growth protocol |
MDA-MB-231 cells were cultured in DMEM-Medium supplemented with 10% FCS (#02.00.830, First Link (UK), 2mM L-Glutamine (#25030-024, Invitrogen), 100U/ml Penicillin and 100μg/ml Streptavidin (#15070-063, Invitrogen) until they were ~60% confluent and then treated for either 48h or 72h with a compound.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using QIAshredder spin columns combined with RNeasy spin-columns. Briefly, medium was removed, cells were lysed and homogenised with 600 of RTL Buffer and then put on a Qiashredder spin column. To the RNA containing flow-through 600of 70% ethanol was added, mixed well and transferred onto RNeasy spin-columns. The columns were treated according to the manufactures protocol and RNA was eluted using 50µRNase free water and immediately stored at -80 °C.
|
| Label |
Cy3
|
| Label protocol |
The samples were labelled with Cy3-labeled CTP fluorescent dyes and the control, untreated total RNA were labelled with Cy5-labeled CTP fluorescent dyes following the Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) v6.5 May 2010 (Agilent Technologies, Palo Alto, CA). The dye-incorporation ratio and the cRNA quantity were determined using the Nanodrop spectrophotometer.
|
| |
|
| Channel 2 |
| Source name |
Untreated MDA-MB-231 cultured for 48h
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MDA-MB-231
|
| Treatment protocol |
Cell cultures were treated for either 48h or 72h with an EZH2/G9a inhibitor compound. Compounds were diluted in DMSO at Stocks of 10mM.
|
| Growth protocol |
MDA-MB-231 cells were cultured in DMEM-Medium supplemented with 10% FCS (#02.00.830, First Link (UK), 2mM L-Glutamine (#25030-024, Invitrogen), 100U/ml Penicillin and 100μg/ml Streptavidin (#15070-063, Invitrogen) until they were ~60% confluent and then treated for either 48h or 72h with a compound.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using QIAshredder spin columns combined with RNeasy spin-columns. Briefly, medium was removed, cells were lysed and homogenised with 600 of RTL Buffer and then put on a Qiashredder spin column. To the RNA containing flow-through 600of 70% ethanol was added, mixed well and transferred onto RNeasy spin-columns. The columns were treated according to the manufactures protocol and RNA was eluted using 50µRNase free water and immediately stored at -80 °C.
|
| Label |
Cy5
|
| Label protocol |
The samples were labelled with Cy3-labeled CTP fluorescent dyes and the control, untreated total RNA were labelled with Cy5-labeled CTP fluorescent dyes following the Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) v6.5 May 2010 (Agilent Technologies, Palo Alto, CA). The dye-incorporation ratio and the cRNA quantity were determined using the Nanodrop spectrophotometer.
|
| |
|
| |
| Hybridization protocol |
For hybridization, 825ng of Cy3-labeled and its respective Cy5-labeled cRNA were added to a microarray slide for 17 hours at 65 C at 10 rpm using the Hybridization Oven kit procedure provided by Agilent Technologies. After hybridization, the slides were then washed per Agilent’s SSPE wash protocol as detailed in Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) v6.5 May 2010 (Agilent Technologies, Palo Alto, CA).
|
| Scan protocol |
The microarray slides were scanned at 3 µm, 20bit resolution with a G2505C DNA microarray scanner (Agilent Technologies, Palo Alto, CA). Data were then extracted from images by the Feature Extraction software version 10.7.3.1, protocol GE2_107_Sep09 (Agilent Technologies, Palo Alto, CA).
|
| Data processing |
Cy3/Cy5 ratios were log2-transformed and lowess-normalized
|
| |
|
| Submission date |
Feb 28, 2012 |
| Last update date |
Aug 27, 2013 |
| Contact name |
Ed Curry |
| E-mail(s) |
e.curry@imperial.ac.uk
|
| Organization name |
Imperial College London
|
| Street address |
Hammersmith Hospital, Du Cane Road
|
| City |
London |
| ZIP/Postal code |
W12 0NN |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL14550 |
| Series (1) |
| GSE36131 |
Breast cancer cell lines treated with EZH2 inhibitor compounds |
|
