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 control U266 cells. It is the second 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.
