|
| Status |
Public on Sep 25, 2015 |
| Title |
Nanos2_conditional_overexpression_Germline_stem_cells_treated_with_DMSO_2 |
| Sample type |
RNA |
| |
|
| Source name |
cultured Germline stem cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: germline stem cells
tissue: testis
Sex: male
|
| Treatment protocol |
Nanos2 cOE GSCs were cultured with DMSO(control) or Tamoxifen (overexpression) for 48 hours. Feeder cells were removed before total RNA extraction.
|
| Growth protocol |
Mouse embryonic fibroblasts (MEFs) derived from C57Bl6 embryos (isolated at day 13.5 post-coitum) were used as feeders for GSC culture. MEF cells treated with mitomycin-C were plated on 0.1% gelatin-coated dishes before GSCs culture. GSCs medium was as previously described. Major growth factors are as followed: rat GDNF (20ng/ml, R&D systems), mouse EGF (20ng/ml, BD Bioscience) and human FGF2 (10ng/ml, Life Technology). For inducing CRE (Rose-CRE-ERT2 knockin mouse) activity in GSCs, 4-Hydroxytamoxifen was added in GSC medium for 48hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with RNeasy mini kit (QIAGEN, Valencia, CA) according to the manufacture's protocol.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.20 µg RNA using the One-Color Low RNA Input 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.
|
| |
|
| Hybridization protocol |
1.5 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 50 µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 50 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Mouse Genome Oligo Microarrays (G4122F) 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.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
gene expression after 48hours incubation with DMSO or Tamoxifen
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.5 (Agilent) using default parameters (protocol GE1-v5_95 and Grid:014868_D_20070207) 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 |
Sep 26, 2014 |
| Last update date |
Sep 25, 2015 |
| Contact name |
ZHI ZHOU |
| E-mail(s) |
zhzhou@nig.ac.jp
|
| Phone |
055-981-6832
|
| Organization name |
National Institute of Genetics, Japan
|
| Department |
Division of Mammalian Development
|
| Lab |
Mammalian Development laboratory
|
| Street address |
Yata 1111
|
| City |
Mishima |
| ZIP/Postal code |
4118540 |
| Country |
Japan |
| |
|
| Platform ID |
GPL7202 |
| Series (1) |
| GSE61808 |
Nanos2-regulated mRNAs in the cultured spermatogonial stem cells (SSCs) |
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