|
| Status |
Public on Apr 03, 2012 |
| Title |
LPS-StimB_flfl_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
LPS-StimB_flfl_rep1
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
tissue: spleen
cell type: B cell
genotype/variation: Ebf1fl/flRERTCre
tamoxifen-citrate treatment: yes
lps stimulation: yes
|
| Treatment protocol |
Ebf1 fl/fl-RERT-Cre mice and heterozygote control animals were treated with 3mg Tamoxifen-Citrate on four consecutive days and were sacrificed for analysis on the tenth day after the start of the experiment. B220+AA4.1- and B220+AA4.1-CD21lo splenic B cells were sorted from Tamoxifen-treated Ebf1+/flRERTCre and Ebf1fl/flRERTCre mice in triplicates, and total RNA was extracted.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared from sorted mouse splenic B cells using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 ug RNA using the Quick Amp Labeling Kit (One-Colo)r and RNA Spike-In Kit, One-Color (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN). 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 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 Human Genome Oligo Microarrays (G4112A) 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 after conditional deletion of Ebf1 following LPS-stimulation
Sample name: p2052LPS
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) 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 |
Feb 24, 2012 |
| Last update date |
Apr 03, 2012 |
| Contact name |
Soeren Boller |
| E-mail(s) |
boller@ie-freiburg.mpg.de
|
| Phone |
0049(0)7615108755
|
| Organization name |
Max Planck Institute of Immunobiology and Epigenetics
|
| Department |
Cellular & Molecular Immunology
|
| Lab |
Grosschedl lab
|
| Street address |
Stuebeweg 51
|
| City |
Freiburg |
| ZIP/Postal code |
79108 |
| Country |
Germany |
| |
|
| Platform ID |
GPL7202 |
| Series (1) |
| GSE36061 |
Microarray analysis to identify genes that are differentially expressed in resting and in LPS-activated splenic B cells of Ebf1+/fl-RERTCre and Ebf1fl/fl-RERTCre mice |
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