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| Status |
Public on Oct 19, 2006 |
| Title |
Transcriptional profile of Candida glabrata ingested by macrophage cells for 2 hours (Experiment 3-1) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
C. glabrata ingested by murine macrophage-like cells J774A.1 for 2 hours
|
| Organism |
Nakaseomyces glabratus |
| Characteristics |
Murine macrophage-like cell line J774A.1 was purchased from ATCC and it is routinely cultured at 37 C in humidified 5 % CO2 atmosphere in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS).
A wild type strain of C. glabrata, Cg462 was used in this experiment and it is routinely cultured in YPD medium at 30 C.
|
| Treatment protocol |
J774A.1 macrophage-like cells were seeded in a 175 cm2 tissue culture flasks and cultured for 16-18 hrs till confluency. Log phase C. glabrata cells (about 2*107 cells/flask) were added onto the macrophage cells to a MOI of 1:1. The flask was spun down for 1 min at 1,000 rpm to bring the yeast cells down. The flask was incubated at 37 C. After 1 hr, the yeast cells that remained outside of macrophages were washed off with warm PBS, and fresh medium was added. At hour 2, the macrophages were lysed in ice-cold water containing RNase cocktail (Ambion Biotech)(to degrade the released macrophage RNA) for 5 min. The lysate was quickly spun down, washed twice with RNase-free water containing protectRNA TM RNase inhibitor (Sigma). Finally the yeast cell pellets from 6 flasks (1*108 cells total) was quickly frozen in liquid nitrogen for RNA prep.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
C. glabrata cells were disrupted with 0.5-mm glass beads (Fisher Scientific) in the presence of guanidium-isothiocyanate (GITC), and the total RNA was isolated by acid phenol extraction. The RNA was further purified by RNeasy Mini kit (Qiagen).
|
| Label |
Cy5-cDNA
|
| Label protocol |
20 ug of total RNA was used to synthesize Cy5-cDNA probes with an anchor primer [(dT)18(dA/dG/dC)] in the presence of Cy5-dCTP (PerkinElmer) using SuperscriptTM III reverse transcriptase (Invitrogen). The probe was purified using MinElute TM reaction Cleanup kit (Qiagen) and vaccum dried.
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| |
|
| Channel 2 |
| Source name |
C. glabrata medium control
|
| Organism |
Nakaseomyces glabratus |
| Characteristics |
see in channel 1
|
| Treatment protocol |
C. glarata cells cultured in DMEM supplemented with 10 % FBS for 2 hrs.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
see in channel 1
|
| Label |
Cy3-cDNA
|
| Label protocol |
see in channel 1
|
| |
|
| |
| Hybridization protocol |
Both Cy5- and Cy3- cDNA probes were resuspened into hybridization buffer (35% formamide, 5*SSC, 0.1%SDS, 0.1 mg/ml denatured salmon sperm DNA) and combined to a total volume of 40 ul. The probe mix was denatured at 100 C for 10 s and quickly cooled on ice, then it was added on a microarray slides. The slides was covered with a cover glass and incubated in a chamber (Corning) at 42 C for 16-18 hrs. The washing protocol follows the manual for UltraGAPS coated slides (Corning).
|
| Scan protocol |
Hybridized slides was scaned by Axon 4000B microarray scanner with 100% power and PMT ranging 600-800. Data were acquired by Axon GenePix 5.0 software.
|
| Description |
This is experiment 1 of a dye-swap experiment. Experiment 2 is deposited in GSM140626.
|
| Data processing |
The median signal intensity captured from Cy5-channel (F635) and from Cy3-channel (F532) was used to calculate the C. glabrata gene expression ratio (Macrophage ingested/medium control
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| |
|
| Submission date |
Oct 17, 2006 |
| Last update date |
Oct 18, 2006 |
| Contact name |
Brendan Cormack |
| E-mail(s) |
bcormack@jhmi.edu
|
| Phone |
410-955-3651
|
| Organization name |
Johns Hopkins Univ. School of Medicine
|
| Department |
Molecular Biology & Genetics
|
| Street address |
Hunterian Building 617, 725 N. Wolfe St.
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21205 |
| Country |
USA |
| |
|
| Platform ID |
GPL3922 |
| Series (1) |
| GSE6058 |
Transcriptional profile of Candida glabrata ingested by murine macrophage-like cells (J774A.1) for 2 and 6 hours |
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