|
| Status |
Public on Jan 18, 2015 |
| Title |
MEF_XBP1WT_Tun_24hr_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
mouse embryonic fibroblast, XBP1WT, Tun, 24hr, rep2
|
| Organism |
Mus musculus |
| Characteristics |
cell type: embryonic fibroblast
background strain: C57BL/6
genotype: XBP1WT
treatment: tunicamycin
time: 24hr
|
| Treatment protocol |
MEFs were treated with DMEM supplemented with 10% fetal bovine serum, 55uM 2-mercaptoethanol, 1x MEM non-essential amino acids, 100units/ml penicillin and 100ug/ml streptomycin containing 2ug/mL tunicamycin for 12 or 24 hours at 37 ºC in a humidified incubator with 5% CO2.
|
| Growth protocol |
MEFs were cultured in DMEM supplemented with 10% fetal bovine serum, 55uM 2-mercaptoethanol, 1x MEM non-essential amino acids, 100units/ml penicillin and 100ug/ml streptomycin at 37 ºC in a humidified incubator with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
total RNA was prepared using the mirVana miRNA Isolation Kit (Ambion, Life Technologies, Carlsbad, CA) following the manufacturer's recommendations. RNA was quantified using a NanoDrop spectrophotometer and quality was monitored by agarose gel electrophoresis of RNA under denaturing conditions.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 75 ng RNA using the Low Input Quick Amp Labeling Kit (One-Color) (Agilent) according to the manufacturer's instructions, followed by RNeasy mini column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop Spectrophotometer.
|
| |
|
| Hybridization protocol |
600ng 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 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 GE Hybridization Buffer HI-RPM was added to the fragmentation mixture and hybridized to Agilent Whole Mouse Genome Oligo Microarrays (G4852A) for 17.5 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 air-dried immediately.
|
| Scan protocol |
Slides were scanned immediately after washing and drying on the Agilent DNA Microarray Scanner (G2505C) 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%, NoXDR setting).
|
| Description |
Gene expression of MEF (XBP1WT) after 24hr treatment of 2ug/mL tunicamycin
Raw data file: 101202_1_2_XBP1WT_Tun24hr_MEF_2.txt
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 028005_D_F_20100422) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Jan 18, 2012 |
| Last update date |
Jan 18, 2015 |
| Contact name |
Seiichi Oyadomari |
| E-mail(s) |
oyadomar@genome.tokushima-u.ac.jp
|
| Phone |
81-88-633-9450
|
| Organization name |
Institute for Genome Research
|
| Department |
Division of Molecular Biology
|
| Street address |
3-18-15 Kuramoto
|
| City |
Tokushima |
| State/province |
Tokushima |
| ZIP/Postal code |
770-8503 |
| Country |
Japan |
| |
|
| Platform ID |
GPL10787 |
| Series (2) |
| GSE35173 |
Development of mRNA and lincRNA expression signatures of MEFs deficient in ER stress mediators for tunicamycin treatment |
| GSE35209 |
Development of mRNA and miRNA expression signatures of MEFs deficient in ER stress mediators for tunicamycin treatment |
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