|
| Status |
Public on Feb 06, 2013 |
| Title |
GRA16-dependent host transcriptional responses in mouse BMDM infected with T. gondii [Pru_GRA16-KO] |
| Sample type |
RNA |
| |
|
| Source name |
mouse BMDM infected with T. gondii Pru_GRA16-KO
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
host cell: Mouse bone marrow-derived macrophages (BMDM)
parasite: Pruku80(deltaGRA16) mutant
time point: 18 h post-infection
|
| Treatment protocol |
Mouse BMDM were infected with Toxoplasma gondii tachyzoites (prepared from infected HFFs and washed in PBS) of the indicated strain at multiplicity of infection ~3; 18 hours post-infection, adherent cells and cells in the supernatent were harvested and lysed in Trizol reagent for RNA extraction.
|
| Growth protocol |
Bone marrow-derived macrophages were obtained from 8-10-week old C57BL/6 mice purchased from JANVIER (France). Bone marrow
was isolated by flushing hind tibias and femurs using a 25-gauge needle and DMEM media supplemented with 10% heat-inactivated FBS (Invitrogen), 1 mM L-glutamine, 50 µg/mL each of penicillin and streptomycin. ~5 x 10^6 cells were resuspended in DMEM supplemented with 20 ng/mL M-CSF (Invitrogen)and plated in 10-cm nontissue culture-treated dishes (Corning). Cells were incubated at 37°C, with 5% CO2 in humidified air for 6 days. Adherent cells were harvested in cold-PBS(4°C) using a cell scraper, and replated at 8 x 10e6 cells per 10-cm2 plate for the assay.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
|
| Label |
Cy5
|
| Label protocol |
NimbleGen One-Color DNA Labeling Kit was used for sample labeling. Dyes used were Cy5
|
| |
|
| Hybridization protocol |
The sample preparation and microarray hybridization were performed based on the manufacturer’s standard protocols. Briefly, total RNA from each sample was amplified and transcribed into fluorescent cRNA with using the manufacturer’s Agilent’s Quick Amp Labeling protocol (version 5.7, Agilent Technologies). The labeled cRNAs were hybridized onto the Whole Genome Oligo Array (4x44K, Agilent Technologies).
|
| Scan protocol |
After washing, slides were scanned with the Agilent DNA microarray Scanner. Data was extracted and normalized using Agilent Feature Extraction Software (version 11.0.1.1).
|
| Description |
Gene expression data from BMDM infected with indicated T. gondii strain, 18 h post-infection
|
| Data processing |
Quantile normalization and subsequent data processing were performed using the GeneSpring GX v11.5.1 software package (Agilent Technologies). After quantile normalization of the raw data, genes that at least 6 out of 9 samples have flags in Detected or Not Detected (“All Targets Value”) were chosen for further data analysis. Differentially expressed genes with statistical significance were identified through Volcano Plot filtering.
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| |
|
| Submission date |
Jun 12, 2012 |
| Last update date |
Feb 06, 2013 |
| Contact name |
Mohamed-ali HAKIMI |
| E-mail(s) |
[email protected]
|
| Phone |
(33)476637469
|
| Organization name |
CNRS
|
| Department |
UMR5309
|
| Lab |
IAB - HAKIMI Team
|
| Street address |
Domaine de la Merci, Campus Santé
|
| City |
LA TRONCHE |
| State/province |
Grenoble |
| ZIP/Postal code |
38700 |
| Country |
France |
| |
|
| Platform ID |
GPL11202 |
| Series (1) |
| GSE38779 |
Host cell subversion by Toxoplasma GRA16, an exported dense granule protein that targets the host cell nucleus and alters gene expression |
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