|
| Status |
Public on Oct 19, 2020 |
| Title |
A20-GFP 24h Dox 3 |
| Sample type |
RNA |
| |
|
| Source name |
BALB/c B cell lymphoma
|
| Organism |
Mus musculus |
| Characteristics |
cell line: A20-GFP
modification to cells: IRES-EGFP expression construct
treatment: Dox
time point: 24 hours
|
| Treatment protocol |
For induction of A20-GFP, A20-mLANA WT or A20-mLANA 3A, cells were cultured in a T-75 flask at a density of 0.2 x 10^6 cells/ml supplemented with doxycycline at a final concentration of 1 µg/ml.
|
| Growth protocol |
A20-GFP, A20-mLANA WT and A20-mLANA 3A mutant were cultured in RPMI 1640 with 10% (v/v) FCS. All cells were incubated in a humidified incubator with 5% CO2 at 37°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from A20-GFP, A20-mLANA WT and A20-mLANA 3A mutant cells in the absence and presence of 1 µg/ml doxycycline for 24 and 48 hours was isolated using the Qiagen RNeasy® Mini kit.
|
| Label |
Cy3
|
| Label protocol |
Synthesis of Cy3- or Cy5-labeled cRNA was performed with the ‘Quick Amp Labeling kit, two color’ (#5190-0444, Agilent Technologies) according to the manufacturer’s recommendations, except for a two-fold downscaling of all reaction volumes.
|
| |
|
| Hybridization protocol |
cRNA fragmentation, hybridization and washing steps were carried-out as recommended in the ‘Two-Color Microarray-Based Gene Expression Analysis Protocol V5.7’ (Agilent), except that 2000 ng of each labelled cRNA population were used for hybridization.
|
| Scan protocol |
Slides were scanned on the Agilent Micro Array Scanner G2565CA (pixel resolution 5 µm, bit depth 20).
|
| Description |
M3850g
A20 cells transduced with lentivirus containing IRES-EGFP expression construct.
Gene expression 24 hours after Dox treatment.
|
| Data processing |
Data extraction and processing of raw fluorescence intensity values were performed with the ‘Feature Extraction Software V10.7.3.1’ by using the recommended default extraction protocol file: GE2_107_Sep09.xml. Measurements of on-chip replicates were averaged using the geometric mean of ProcessedSignal (PS) values (for each channel independently) to retrieve one resulting value per probe, sample, and channel. Single Features were excluded from averaging, if they i) were manually flagged, ii) were identified as Outliers by the Feature Extraction Software, iii) lay outside the interval of ‘1.42 x interquartile range‘ regarding the PS distribution of the respective on-chip replicate population, or, iv) showed a coefficient of variation of pixel intensities per feature that exceeded 0.5.
For inter-array normalization, averaged processed intensity values, ‘gProcessedSignal’ (gPS) and ‘rProcessedSignal’ (rPS), were further transformed as follows: All PS values (gPS and rPS) of one particular microarray (‘Array i’ in the formula shown below) were multiplied with a microarray-specific scaling factor. This factor was calculated by dividing a ‘reference 75th Percentile value’ (set as 1500 for the whole series) by the 75th Percentile value of the gPS calculated by the Feature Extraction software for that microarray. Accordingly, normalized PS values for all samples (microarray data sets) were calculated by the following formula:
normalized PSArray i = PSArray i x (1500 / 75th Percentile gPSArray i)
Finally, a lower intensity threshold (surrogate value) was defined based on intensity distribution of negative control features. This value was fixed at 15 normalized PS units. All of those measurements that fell below this intensity cutoff were substituted by the respective surrogate value of 15.
|
| |
|
| Submission date |
Jun 25, 2020 |
| Last update date |
Oct 19, 2020 |
| Contact name |
Rishikesh Lotke |
| Organization name |
Medizinische Hochschule Hannover
|
| Department |
Institut für Virologie
|
| Lab |
Thomas F. Schulz
|
| Street address |
Carl-Neuberg-Straße 1
|
| City |
Hannover |
| State/province |
Niedersachsen |
| ZIP/Postal code |
30625 |
| Country |
Germany |
| |
|
| Platform ID |
GPL11202 |
| Series (2) |
| GSE153236 |
Brd/BET Proteins Influence the Genome-Wide Localization of the Kaposi’s Sarcoma-associated Herpesvirus and Murine Gammaherpesvirus Major Latency Proteins [mouse array] |
| GSE153244 |
Brd/BET Proteins Influence the Genome-Wide Localization of the Kaposi’s Sarcoma-associated Herpesvirus and Murine Gammaherpesvirus Major Latency Proteins |
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