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Status |
Public on Aug 27, 2013 |
Title |
3h_USP2KD_treated_2 |
Sample type |
RNA |
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Source name |
differentated 3T3-L1 cells stimulated with supernatant of USP2 knockdown HL-60 cells for 3 hrs
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Organism |
Mus musculus |
Characteristics |
cell line: 3T3-L1 cell type: adipocytes
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Treatment protocol |
After differentiation, 3T3-L1 cells were treated with 2-fold diluted supernatant with supernatants of USP2 knockdown or mock transfected HL-60 cells for 3 or 6 hrs. Supernatants were prepared from 5 day culture of USP2 knockdown or control cells in Opti medium (Invitrogen) with 30nM PMA
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Growth protocol |
3T3-L1 cells were grown in DMEM with 10% FCS. For differentiation into adipocyte-like cells, we cultured the cells in the adipogenic medium (Takara) for 1 week.
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Extracted molecule |
total RNA |
Extraction protocol |
Total cellular RNA was prepared with TRIzol reagent according to a manual.
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Label |
Cy3
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Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.1 ug RNA using the One-Color Low Input Quick Amp WT Labeling 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-2000 Spectrophotometer.
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Hybridization protocol |
0.6 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 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Mouse Genome Oligo Microarrays (G4852A) 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.
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Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
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Description |
3T3-L1 cells stimulated with supernatant of USP2 knockdown HL-60 cells, 3h, biological rep 2
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Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (GE1_107_Sep09 and Grid:028005_D_F_20120201) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. Normalized signal intensity calculated using GeneSpring 12.1GX. Setting is : Advanced Flag Import (Feature is not positive and significant, Not Detected; Feature in not Uniform, Compromised; Feature is not above background, Not Detected; Feature is Saturated, Compromised; Feature is a population outlier, Compromised), Normalization Option (Threshold raw signals to 1.0; Normaliation algorithm, Percentile Shift; Percentile target, 75), Preprocess Baseline Options (Do not perform baseline transformation)
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Submission date |
Jan 07, 2013 |
Last update date |
Aug 27, 2013 |
Contact name |
Hiroshi Kitamura |
E-mail(s) |
ktmr@rakuno.ac.jp
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Phone |
81-11-388-4781
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Organization name |
Rakuno Gakuen University
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Department |
School of Veterinary Medicine
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Lab |
Laboratory for Veterinary Physiology
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Street address |
582 Midorimachi, Bunkyodai
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City |
Ebetsu |
ZIP/Postal code |
069-8501 |
Country |
Japan |
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Platform ID |
GPL10787 |
Series (1) |
GSE43313 |
Effects of supernatants of USP2 knockdown HL-60 cells and mock transfected HL60 cells on differentiated 3T3-L1 cells |
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