|
| Status |
Public on Mar 13, 2014 |
| Title |
CD9 overexpression cells rep-1 |
| Sample type |
RNA |
| |
|
| Source name |
U266 cells with CD9 overexpression
|
| Organism |
Homo sapiens |
| Characteristics |
genotype: overexpression of CD9
|
| Treatment protocol |
Briefly, when the U266 cells reached 50-60% confluence, the medium was removed and washed twice with PBS. Polybrene was used to increase the infection rate, and the infection was performed with lenti-CD9 and lenti-GFP according to the manufacturer’s instructions. At 2 days post-infection, the percentage of GFP-positive U266 cells was determined using a fluorescence microscope to evaluate the infectivity. GFP-positive cells were selected by flow cytometry.
|
| Growth protocol |
RPMI1640 medium with 15% fetal bovine serum, 100 units/mL penicillin and 100 mg/mL streptomycin
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from cell pellets by Trizol (Invitrogen) and reverse transcribed into cDNA using MultiScribe Reverse Transcriptase (Applied Biosystems, Foster city, CA, USA) according to the manufacturer’s instructions
|
| Label |
Cy3
|
| Label protocol |
Briefly, ds-cDNA was incubated with 4 μg RNase A at 37°C for 10 min and cleaned using phenol:chloroform:isoamyl alcohol, followed by ice-cold absolute ethanol precipitation. The purified cDNA was quantified using a NanoDrop ND-1000. For Cy3 labeling of cDNA, the NimbleGen One-Color DNA labeling kit was used according to the manufacturer's guideline detailed in the Gene Expression Analysis protocol (NimbleGen Systems, Inc., Madison, WI, USA)
|
| |
|
| Hybridization protocol |
Microarrays were hybridized at 42°C during 16 to 20h with 4 μg of Cy3 labeled ds-cDNA in NimbleGen hybridization buffer/hybridization component A in a hybridization chamber (Hybridization System - NimbleGen Systems, Inc., Madison, WI, USA). Following hybridization, washing was performed using the NimbleGen Wash Buffer kit (NimbleGen Systems, Inc., Madison, WI, USA).
|
| Scan protocol |
After being washed in an ozone-free environment, the slides were scanned using the Agilent Scanner G2505C.
|
| Description |
This sample is of U266 cells with CD9 overexpression. It is the first of three wild-type biological replicates used in this experiment, each from separate cultures.
|
| Data processing |
Slides were scanned at 5 μm/pixel resolution using an Agilent Scanner G2505C scanner piloted by GenePix Pro 6.0 software (Axon). Scanned images (TIFF format) were then imported into NimbleScan software (version 2.5) for grid alignment and expression data analysis. Expression data were normalized through quantile normalization and the Robust Multichip Average (RMA) algorithm included in the NimbleScan software. The Probe level (*_norm_RMA.pair) files and Gene level (*_RMA.calls) files were generated after normalization. All gene level files were imported into Agilent GeneSpring GX software (version 11.5.1) for further analysis. Differentially expressed genes were identified statistical significance were identified through Volcano Plot filtering. Hierarchical clustering was performed using the Agilent GeneSpring GX software (version 11.5.1). GO analysis and Pathway analysis were performed using the standard enrichment computation method.
|
| |
|
| Submission date |
Mar 12, 2014 |
| Last update date |
Mar 13, 2014 |
| Contact name |
Xiaotong Hu |
| E-mail(s) |
hxt_hangzhou@sina.com
|
| Organization name |
Zhejiang University
|
| Street address |
Qingchun East Road #3
|
| City |
Hangzhou |
| ZIP/Postal code |
310016 |
| Country |
China |
| |
|
| Platform ID |
GPL16025 |
| Series (1) |
| GSE55818 |
Down-regulation of CD9 by methylation decreased bortezomib sensitivity in multiple myeloma |
|
