|
| Status |
Public on Jun 27, 2015 |
| Title |
Nr4a-deficient |
| Sample type |
RNA |
| |
|
| Source name |
Nr4a-deficient Treg cells
|
| Organism |
Mus musculus |
| Characteristics |
age: 5 week old
cell type: regulatory T cells
genotype: Nr4a-deficient
|
| Treatment protocol |
Treg cells from wild-type or Nr4a-TKO mice were sorted, and directly lysed with RNA-iso total RNA extraction solution (Takara, Japan).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the RNA-iso (Takara, Japan) following the manufacturer's recommendations. Extracted total RNA was further purified using RNAeasy-micro kit. 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 cRNA was prepared from 600 ng RNA using the Low Input Quick Amp labeling kit, one-color (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 |
480 ng of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Mouse Genome Oligo Microarray Ver2.0 (4x44k) Microarrays 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 (G2539A) using one color scan setting for 4x44k array slides ( Scan resolution 3 um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression in Nr4a-deficient Treg cells
|
| Data processing |
The scanned images were analyzed with GeneSpring GX Ver.11.0 (Agilent) using default parameters (protocol GE1_1010_sep10 and Grid: 028005_D_F_20130207) 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 |
Jun 26, 2015 |
| Last update date |
Jun 27, 2015 |
| Contact name |
Takashi Sekiya |
| E-mail(s) |
lb-sekiya@hospk.ncgm.go.jp
|
| Organization name |
National Center for Global Health and Medicine
|
| Street address |
1-7-1 Kohnodai
|
| City |
Ichikawa |
| State/province |
Chiba |
| ZIP/Postal code |
272-0827 |
| Country |
Japan |
| |
|
| Platform ID |
GPL11202 |
| Series (1) |
| GSE70306 |
Comparison of mRNA expression pattern between wild-type regulatory T (Treg) cells and Nr4a-deficient Treg cells |
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