|
| Status |
Public on Sep 13, 2012 |
| Title |
IRF4-mut_Th17_cells_42hr_rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
IRF4-mut_Th17_cells_42hr
|
| Organism |
Mus musculus |
| Characteristics |
strain background: C57BL/6J
genotype/variation: IRF4 -/-
tissue: spleen
cell type: Naïve CD4+, CD62L hi, CD25 - T cells
differentiation condition: Th17
timepoint: 42 hrs
|
| Growth protocol |
Naïve T- cells isolated from spleen were cultured on a-CD3 coated plates with 2.5 mg/ml a-CD28, 20 ng/ml IL-6, 20 ng/ml IL-23, 1 ng/ml hTGFb, 10 mg/ml a-IL4 and 10 mg/ml a-IFNg for 42 hrs
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Qiagen RNeasy kit following manufacturer's protocol and including on-column DNase-digestion. RNA was quantified using UV-spec Nanodrop and then profiled on Agilent Bioanalyzer.
|
| Label |
Cy5
|
| Label protocol |
Total RNA was converted to double-stranded cDNA and then into Cy-dye labeled cRNA using Agilent’s Quick Amp Labeling Kit. 750 ng of the labeled cRNA was fragmented and hybridized against Cy3-labeled Universal mouse reference (Stratagene, La Jolla, CA).
|
| |
|
| Channel 2 |
| Source name |
Stratagene's Universal Mouse Reference RNA (UMRR) cat # 740100
|
| Organism |
Mus musculus |
| Characteristics |
sample type: Reference: pooled RNA from 11 tissues
|
| Growth protocol |
Naïve T- cells isolated from spleen were cultured on a-CD3 coated plates with 2.5 mg/ml a-CD28, 20 ng/ml IL-6, 20 ng/ml IL-23, 1 ng/ml hTGFb, 10 mg/ml a-IL4 and 10 mg/ml a-IFNg for 42 hrs
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Qiagen RNeasy kit following manufacturer's protocol and including on-column DNase-digestion. RNA was quantified using UV-spec Nanodrop and then profiled on Agilent Bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
Total RNA was converted to double-stranded cDNA and then into Cy-dye labeled cRNA using Agilent’s Quick Amp Labeling Kit. 750 ng of the labeled cRNA was fragmented and hybridized against Cy3-labeled Universal mouse reference (Stratagene, La Jolla, CA).
|
| |
|
| |
| Hybridization protocol |
Agilent In Situ Hybridization Kit Plus was used where Cy-dye labeled control and test samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers. After hybridization, the arrays were washed twice, dried and then scanned.
|
| Scan protocol |
Scanned on an Agilent scanner, images were processed using Agilent Feature Extraction software version 10.7.
|
| Description |
IRF4 -/- rep1
|
| Data processing |
The matrix data table was created by first log transforming the raw data and then filtering out all probes that showed a standard deviation of <0.25 across all samples as well as probes that do not have an associated entrez id. Agilent Feature Extraction software version 10.7 was used for background subtraction and LOWESS normalization.
The normalized data was then analyzed further using Partek Genomic Suite Version 6.6 by performing a 1-way ANOVA test between the IRF4 KO vs IRF4 Het samples. Data for the probes which showed a > 2-fold change (up or down) with a p-value <0.05 is presented in the 'Analyzed_data.txt' file (available on Series records). It is presented in ascending order of differential expression (Fold-Change) between the IRF4 KO vs IRF4 Het samples. The probeset ID, Gene Symbol, p-value of differential expression and Fold-Change between the IRF4 KO and IRF4 Het samples are indicated. Negative fold changes indicate down-regulation in IRF4 KO samples and positive fold changes indicate up-regulation.
|
| |
|
| Submission date |
Sep 10, 2012 |
| Last update date |
Sep 13, 2012 |
| Contact name |
Smita Agrawal |
| Organization name |
Genentech Inc.
|
| Department |
Immunology
|
| Street address |
1 DNA Way
|
| City |
South San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94080 |
| Country |
USA |
| |
|
| Platform ID |
GPL7202 |
| Series (2) |
| GSE40483 |
A genomic regulatory element that directs assembly and function of immunespecific AP-1-IRF complexes |
| GSE40726 |
Transcriptional profiling of IRF4 -/- vs IRF4 +/- T-cells under Th17 polarizing conditions |
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