|
| Status |
Public on May 25, 2011 |
| Title |
GCN5A-KO_control_3 |
| Sample type |
RNA |
| |
|
| Source name |
T.Gondii
|
| Organism |
Toxoplasma gondii |
| Characteristics |
genotype: GCN5A KO
treatment: control
|
| Treatment protocol |
Tachyzoites were released from HFF monolayers using a 25 gauge syringe needle, and filtered to remove the host cell debris was spun out at 500 ×g for 5 min. Extracellular parasites were resuspended at a concentration of 105 parasites/ml in DMEM or alkaline medium (pH 8.2, adjusted with NaOH). This suspension was incubated at 37°C in 5% CO2 for 30 min, and then 103 parasites were inoculated onto HFF monolayers in 24-well plates. After 5 day incubation at 37°C, the infected monolayers harvested for RNA isolation.
|
| Growth protocol |
Toxoplasma tachyzoites (wild-type (WT) RH and deltaGCN5-A lines) were cultured in primary human foreskin fibroblasts (HFF) using Dulbecco’s Modified Eagle’s Medium supplemented with 1.0% fetal bovine serum (Invitrogen). Parasites were grown in a humidified CO2 (5%) incubator at 37°C. Cultures were confirmed to be free of Mycoplasma contamination by MycoAlertTM Assay (Cambrex Bio Science). Parasites were harvested immediately following lysis of host cell monolayers and purified by filtration through a 3.0 micron filter.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from freshly lysed, filter-purified parasites using an RNeasy Mini Kit according to the manufacturer’s instructions (Qiagen). To minimize genomic DNA contamination, additional treatment with DNase was performed.
|
| Label |
biotin
|
| Label protocol |
Affymetrix standard 3' IVT protocol using 2ug of total RNA.
|
| |
|
| Hybridization protocol |
Following fragmentation, 10 ug of cRNA were hybridized for 17 hr at 45C on Toxoplasma Gondii arrays. GeneChips were washed and stained in the Affymetrix Fluidics Station.
|
| Scan protocol |
GeneChips were scanned using the standard Affymetrix protocol.
|
| Description |
Gene expression data from T.Gondii
|
| Data processing |
The data were analyzed with Affymetrix Gene Chip Operating Software (GCOS) using the MAS 5.0 algorithm using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 1000.
|
| |
|
| Submission date |
Jun 02, 2010 |
| Last update date |
Jan 24, 2013 |
| Contact name |
William J Sullivan |
| E-mail(s) |
[email protected]
|
| Organization name |
Indiana University
|
| Department |
Pharmacology & Toxicology
|
| Street address |
635 Barnhill Dr.
|
| City |
Indiananpolis |
| State/province |
IN |
| ZIP/Postal code |
46202 |
| Country |
USA |
| |
|
| Platform ID |
GPL16542 |
| Series (1) |
| GSE22100 |
Toxoplasma gondii lysine acetyltransferase GCN5-A functions in the cellular response to alkaline stress and expression of cyst genes |
|
