|
| Status |
Public on Nov 30, 2012 |
| Title |
Meis-Low_TEC_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Meis-Low
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
genotype/variation: Meis1-EGFP BAC-transgenic [Tg(Meis1-EGFP)FO156Gsat/Mmcd]
age: 4 weeks
meis1 expression levels: Low
cell type: CD45- EpCAM+ thymic epithelial cell (TEC)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Three populations of CD45- EpCAM+ thymic epithelial cells with different EGFP expression levels and cell sizes (EGFPhigh FSClow, EGFPint FSChigh and EGFP- FSClow) was sorted with a FACSAreaTMII (BD Bioscience, San Diego, CA) from 4-week-old Meis1-EGFP BAC-transgenic reporter mice, Tg(Meis1-EGFP)FO156Gsat/Mmcd), that were generated by the GENSAT BAC transgenic project. RNA was isolated using the Qiagen RNeasy micro kit (Qiagen) following the manufacturer's recommendations. The protocol includes differential lysis of cells, and an on-column DNase digestion. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cDNA was prepared from 10ng RNA using the WT-Ovation™ Pico RNA Amplification System (NuGEN) and Genomic Enzymatic Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cDNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
3 ug of Cy3 labeled cDNA (specific activity >15.0 pmol Cy3/ug cDNA) was 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 by brief centrifugation.
|
| 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 in mouse thymic epithelial cells expresing low levels of Meis1
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.5.3.1 (Agilent) using default parameters (protocol: GE1-v5_95_Feb07 and Grid: 014868_D_F_20100123) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. Normalized data were analyzed using GeneSpring GX version 11.0.2 software (Agilent Technologies, Santa Clara, CA). Threshold raw signals to 1.0, Normalization algorithm: Percentile Shift, Normalization: Shift to 50 percentile
|
| |
|
| Submission date |
Jul 20, 2011 |
| Last update date |
Nov 30, 2012 |
| Contact name |
Ryo Goitsuka |
| E-mail(s) |
[email protected]
|
| Phone |
+81-4-7121-4102
|
| Fax |
+81-4-7121-4103
|
| Organization name |
Research Institute for Biological Sciences
|
| Department |
Division of Development & Aging
|
| Lab |
Goitsuka Laboratory
|
| Street address |
2669 Yamazaki
|
| City |
Noda-shi |
| State/province |
Chiba |
| ZIP/Postal code |
279-0022 |
| Country |
Japan |
| |
|
| Platform ID |
GPL7202 |
| Series (2) |
| GSE30825 |
Transcriptional signatures of thymic epithelial cells with distinct Meis1 expression levels |
| GSE30826 |
The role of Meis1 in the maintenance of postnatal thymic epithelial cells |
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