|
| Status |
Public on Jul 30, 2013 |
| Title |
L428+MAT_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Hodgkin lymphoma cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: L428
treatment: Mifepristone + 5-aza-2′-deoxycytidine + Trichostatin A (MAT)
duration of treatment: 5 days
|
| Treatment protocol |
PAX5 expression was induced in the cell line L428-PAX5 prior to the treatment with 5-aza-2′-deoxycytidine (5-aza-dC; Sigma-Aldrich, St. Louis, MO, USA), a DNA demethylating reagent and Trichostatin A (TSA; Sigma-Aldrich), a histone deacetylase inhibitor. After 2 days of incubation with 0.25 nM mifepristone, the medium was partially replaced and only the freshly added medium was supplemented with mifepristone. 5-aza-dC was subsequently added at a concentration of 3 µM and the cells were incubated for further 3 days. TSA was added at concentration 640 nM 24 h prior to harvesting.
|
| Growth protocol |
The Burkitt lymphoma cell lines Raji and Namalwa (designated as B-cell lines throughout the manuscript) and the Hodgkin cell line L428 were grown in RPMI 1640 (PAA Laboratories) supplemented with 10% fetal calf serum (PAN-Biotech). For the cultivation of the cell line L428-PAX5, 25 mM HEPES (Life Technologies/Invitrogen) was added supplementary. The cell lines were incubated at 37 °C and 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated as described by Ehlers et al.(2008).
|
| Label |
biotin
|
| Label protocol |
Single strand cDNA was prepared from 250 ng total RNA according to the standard Ambion protocol (WT Expression kit) and fragmentation and biotin labeling was done according to the standard Affymetrix protocol (WT terminal labeling kit)
|
| |
|
| Hybridization protocol |
2.5 ug of ss cDNA were hybridized for 16 hr at 45°C on Human Gene ST 1.0 Arrays (Affymetrix). Arrays were washed and stained with standard protocols in the Affymetrix Fluidics Station 450.
|
| Scan protocol |
GeneChips were scanned using the Scanner G3000
|
| Data processing |
Affymetrix CEL files were imported into Partek Genomic Suite Software (Version 6.5, Partek Inc., St. Louis, USA) and processed by the implemented RMA workflow (median polish probe set summarization, RMA background correction, quantile normalization).
probe group file: HuGene-1_0-st-v1.r4.pgf
meta-probeset file: HuGene-1_0-st-v1.r4.mps
|
| |
|
| Submission date |
Feb 11, 2013 |
| Last update date |
Jul 31, 2013 |
| Contact name |
Lora Dimitrova |
| E-mail(s) |
[email protected]
|
| Organization name |
Charité-University Medicine Berlin
|
| Department |
Institute of Pathology
|
| Street address |
Hindenburgdamm 30
|
| City |
Berlin |
| State/province |
Berlin |
| ZIP/Postal code |
12200 |
| Country |
Germany |
| |
|
| Platform ID |
GPL6244 |
| Series (1) |
| GSE44244 |
PAX5 overexpression is not enough to reestablish the mature B-cell phenotype in classical Hodgkin lymphoma |
|
