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Status |
Public on Dec 22, 2020 |
Title |
scribKD_Tet(–)_30hr |
Sample type |
RNA |
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Source name |
scribKD, 30hr, without Tet
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Organism |
Canis lupus familiaris |
Characteristics |
cell line: Madin-Darby Canine Kidney (MDCK) cellls treatment condition: cultured without tetracycline for 30 hours
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Treatment protocol |
The cells were treated with tetracycline at a final concentration of 5 µg/ml at 6 hours after plating.
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Growth protocol |
MDCK-II scribKD cells were plated in a 12-well plate at a density of 154,000 cells/wel. The cells were cultured in DMEM high glucose supplemented with 10% FBS and 100 units/ml penicillin G in a 5% CO2 atmosphere at 37℃.
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Extracted molecule |
total RNA |
Extraction protocol |
At the indicated time after tetracycline addtion, total RNA was extracted from three independent wells of the same condition and mixed using Isogen (Wako) according to the manufacturer's instructions.
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Label |
Cy3
|
Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared using the Low Input Quick Amp Labeling kit (Agilent) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-2000 Spectrophotometer.
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Hybridization protocol |
Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 25 µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Canine (V2) Gene Expression Microarray (Agilent, G2519F-021193) 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).
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Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner using one color scan setting for 4x44k array slides. Dye channel is set to Green and Green PMT is set to 100%.
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Description |
Gene expression at 30 hours after cultured without tetracycline
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Data processing |
The scanned images were analyzed with Feature Extraction Software 11.5.1.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities.
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Submission date |
Jun 24, 2020 |
Last update date |
Dec 22, 2020 |
Contact name |
Motoyuki Ogawa |
Organization name |
The University of Tokyo
|
Department |
Graduate School of Pharmaceutical Sciences
|
Lab |
Cell Signaling
|
Street address |
7-3-1, Hongo
|
City |
Bunkyo-ku |
State/province |
Tokyo |
ZIP/Postal code |
113-0033 |
Country |
Japan |
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Platform ID |
GPL13605 |
Series (1) |
GSE153186 |
Gene expression change of MDCK cells expressing scribble shRNA in a tetracycline-inducible manner (scribKD cells) |
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