|
| Status |
Public on May 31, 2013 |
| Title |
WT8S |
| Sample type |
RNA |
| |
|
| Source name |
primary mouse embryonic fibroblasts (MEFs) from E10.0 embryo obtained from Rb-ATM double knockout C57BL/6J mouse
|
| Organism |
Mus musculus |
| Characteristics |
background strain: C57BL/6J
cell type: primary mouse embryonic fibroblasts (MEFs)
transfection: vector expressing ATM
genotype/variation: Rb-/-;ATM-/-;ATM-reconstituted
|
| Treatment protocol |
Primary MEFs were transduced with empty vector or vector expressing ATM or vector expressing DNMT1 shRNA, selected with 4μg/ml puromycin for 3 days, grown under normal culture conditions for another 4 days and analysed.
|
| Growth protocol |
Primary MEFs were grown in MEM-α supplemented with 10% FBS under normal culture conditions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA isolation RNA was quantified and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng of total RNA using the One-Color Low Input Quick Amp Labeling kit (Agilent) according to the manufacturer's instructions with some changes. 3rd Spike mix was made by diluted 2nd Spike-Mix 10 times, and 1.8 ul of 3rd Spike-Mix was added to samples. Cy3 labeled cRNA was synthesized at 40°C for 20 hours and purified by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.65 ug of Cy3-labelled cRNA (specific activity >7.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Mouse Whole Genome Microarray 4x44K (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 by raise it slowly from buffer 2.
|
| 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 5um, Dye channel is set to Green. Scan mode is eXtended Dynamic range Scan mode and PMT is set to 10% and 100%).
|
| Description |
163 cells reconstituted with vector expressing ATM, selected with 4μg/ml puromycin for 3 days, grown under normal culture conditions for another 4 days and analysed.
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 014868_D_F_20100617) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
May 30, 2013 |
| Last update date |
May 31, 2013 |
| Contact name |
Awad Shamma |
| E-mail(s) |
shamawad@staff.kanazawa-u.ac.jp
|
| Phone |
+81-76-264-6751
|
| Fax |
+81-76-234-4521
|
| URL |
http://omb.w3.kanazawa-u.ac.jp/omb/Home.html
|
| Organization name |
Kanazawa Uiniversity
|
| Department |
Cancer Research Institute
|
| Lab |
Division of Oncology and Molecular Biology
|
| Street address |
Kakuma-machi
|
| City |
Kanazawa |
| State/province |
Ishikawa |
| ZIP/Postal code |
920-1192 |
| Country |
Japan |
| |
|
| Platform ID |
GPL10333 |
| Series (1) |
| GSE47501 |
ATM mediates pRB function to control DNMT1 protein stability and DNA methylation |
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