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Sample GSM1514382 Query DataSets for GSM1514382
Status Public on Sep 25, 2015
Title Nanos2_conditional_overexpression_Germline_stem_cells_treated_with_4-Tamoxifen_1
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.
 
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)

Data table header descriptions
ID_REF
VALUE 75 percentile normalized signal intensity

Data table
ID_REF VALUE
A_51_P378699 8.836589
A_51_P306247 4.9033766
A_51_P306243 2.244176
A_52_P400967 9.904222
A_52_P690656 2.0925226
A_52_P426692 11.397706
A_51_P250993 2.4248004
A_52_P223473 2.277224
A_52_P487599 9.807962
A_52_P487598 8.28484
A_52_P426698 8.91947
A_52_P1196439 2.264205
A_52_P170486 6.571665
A_52_P221696 8.396243
A_52_P1011921 2.3133829
A_51_P420726 8.943092
A_51_P370315 10.101776
A_51_P189733 8.195467
A_52_P659417 12.559175
A_51_P160824 8.757814

Total number of rows: 41174

Table truncated, full table size 906 Kbytes.




Supplementary file Size Download File type/resource
GSM1514382_Nanos2_conditional_overexpression_Germline_stem_cells_Tamoxifen_1.txt.gz 9.2 Mb (ftp)(http) TXT
Processed data included within Sample table

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