|
| Status |
Public on Oct 15, 2021 |
| Title |
KMH2-48H |
| Sample type |
RNA |
| |
|
| Source name |
KMH2 cells
|
| Organism |
Homo sapiens |
| Characteristics |
agent: Ruxolitinib
time point: 48 hours
|
| Treatment protocol |
Cell lines were incubated with or without ruxolitinib (HY-50856, MedChem, Monmouth Junction, NJ).
|
| Growth protocol |
Hodgkin lymphoma cell lines were growth in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum. The cultures were incubated for 6 or 24 hours at 37 ºC in a humidified incubator with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
200 ng of total RNA was isolated using the Recover All kit (ThermoFisher) following the manufacturer's recommendations.
|
| Label |
Cy3
|
| Label protocol |
Whole-transcriptome expression assays were performed using SurePrint G3 Human Gene Expression arrays 8×60K v2 (Agilent Technologies, Santa Clara, CA), profiling approximately 32,000 transcripts. Briefly, 200 ng of total RNA was isolated from all the untreated cell lines and after 6, 12, 24 and 48 h of drug incubation with ruxolitinib at the IC50 doses. Reverse transcription and cDNA labeling with Cy3-CTP were carried out according to the manufacturer’s instructions.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) 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), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
48 hours of Ruxolitinib treatment
|
| Data processing |
Scanning, quantification, and data normalization were done using a G4900DA SureScan Microarray Scanner System and Feature Extraction v12.0 (Agilent Technologies). The extracted data were normalized and analyzed with the Subió platform (Subió Inc.). The normalization of one-color arrays was the Percentile Shift system.
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| |
|
| Submission date |
Oct 13, 2021 |
| Last update date |
Oct 16, 2021 |
| Contact name |
Sara Fernández Robledo |
| E-mail(s) |
sardelf@hotmail.com
|
| Organization name |
Fundación MD Anderson
|
| Street address |
C/ Arturo Soria, 270
|
| City |
Madrid |
| State/province |
Madrid |
| ZIP/Postal code |
28033 |
| Country |
Spain |
| |
|
| Platform ID |
GPL17077 |
| Series (1) |
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