|
| Status |
Public on Sep 25, 2016 |
| Title |
RNA from Met-high cells of B16-F10 cell line_1 |
| Sample type |
RNA |
| |
|
| Source name |
B16-F10, Met-high cells, replicate1
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Skin
cell line: B16-F10 malignant melanoma cells
sorted cells: Met-high cells
|
| Treatment protocol |
B16-F10 cell were individually sorted and divided into Met-low or Met-high population using a FACS Aria II. Sorted cells were cultured for 3 days, independently.
|
| Growth protocol |
B16-F10 cell was cultured with RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum. The cultures were incubated at 37 ºC in a humidified incubator with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared using Sepazol reagent (Nakarai Tesque). The quality of RNA samples were assessed by RNA 6000 nano kit (Agilent Bioanalyzer 2100).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng RNA using the Low RNA Input Fluorescent Linear Amplification kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
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| |
|
| Hybridization protocol |
1.65 ug of Cy3-labelled cRNA (specific activity >6.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Mouse Genome Microarray 4x44K (G4846A) 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 by raise it slowly from buffer 2.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565BA) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green. Scan mode is eXtended Dynamic range Scan mode and PMT is set to 10% and 100%).
|
| Description |
Gene Expression in Met-high cells of B16-F10 melanoma
Met-high_1
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 026655_D_20120201) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
Jun 10, 2015 |
| Last update date |
Sep 26, 2016 |
| Contact name |
Kunio Matsumoto |
| E-mail(s) |
[email protected]
|
| Organization name |
Kanaawa University
|
| Street address |
Kakuma
|
| City |
Kanazawa |
| ZIP/Postal code |
920-1192 |
| Country |
Japan |
| |
|
| Platform ID |
GPL11202 |
| Series (1) |
| GSE69741 |
Expression data of Met-low and Met-high B16-F10 malignant melanoma cells |
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