|
| Status |
Public on Dec 01, 2013 |
| Title |
4 day biofilm + 4 hr neutrophil biological replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
S. aureus 4 day biofilm exposed to neutrophils for 4 hr
|
| Organism |
Staphylococcus aureus |
| Characteristics |
sample: Whole cell
biofilm: 4 day
exposure time: 4 hr
exposure item: Neutrophils
|
| Treatment protocol |
Following biofilm formation macrophages or neutrophils (10^7 and 10^6, respectively) were co-cultured with S. aureus biofilms at 37C for the indicated time period to assess their impact on the biofilm transcriptome. Mock treatment refers to the addition of RPMI media.
|
| Growth protocol |
Sterile 2-well glass chamber slides (Nunc, Rochester, NY) were treated with 20% human plasma overnight at 4C. The following day, plasma coating buffer was removed and each chamber inoculated with S. aureus strain USA300 LAC and incubated at 37C under static conditions for a period of 4 or 6 days to gnerate immature and mature biofilms, respectively. Medium was replenished every 24 h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Biofilm media was removed and replaced with RNAprotect (Qiagen, Hilden, Germany). Biofilms were collected from the bottom of the chamber slide using a cell scraper, sonicated for 5 min, then pelleted by centrifugation for 5 min. Cell pellets were resuspended in RLT buffer (Qiagen), transferred to a 2 ml FastPrep lysing tube (MP Biomedicals, Santa Ana, CA) and then lysed in a FastPrep homogenizer (MP Biomedicals) for 20 sec on speed setting 6. Cell debri was collected by centrifugation. RNA was isolated from the supernatant using RNeasy mini kits (Qiagen) and contaminating DNA was removed by on-column DNase digestion using RNase-Free DNase (Qiagen).
|
| Label |
Biotin
|
| Label protocol |
Total bacterial RNA (75 ng) isolated from each sample was independently amplified using ExpressArt Bacterial mRNA amplification Nano kits (AmpTec GmbH, Germany) and biotinylated using BioArray HighYield RN Transcript Labeling kits (Enzo Life Sciences, Inc., Farmingdale, NY).
|
| |
|
| Hybridization protocol |
3 ug of labeled RNA were hybrized for 16 hr at 45C on a S. aureus Affymetrix GeneChip, following the manufacturer's recommendations for antisense prokaryotic arrays (Affymetrix, Santa Clara, CA). GeneChips were washed and stained in an Affymetrix Fluidics Station using ProKaryotic GE-wash2 protocol.
|
| Scan protocol |
GeneChips were scanned in an Affymetrix 7G GeneArray Scanner
|
| Description |
Gene expression data from 4 day biofilms exposed to Neutrophils for 4 hr
|
| Data processing |
The data were analyzed with Microarray Suite version 5.0 using Affymetrix default analysis settings.
|
| |
|
| Submission date |
Sep 06, 2013 |
| Last update date |
Dec 01, 2013 |
| Contact name |
Paul Dunman |
| E-mail(s) |
[email protected]
|
| Phone |
585-276-5700
|
| Organization name |
University of Rochester
|
| Department |
Microbiology and Immunology
|
| Street address |
601 Elmwood Avenue
|
| City |
Rochester |
| State/province |
NY |
| ZIP/Postal code |
14642 |
| Country |
USA |
| |
|
| Platform ID |
GPL1339 |
| Series (1) |
| GSE50675 |
Global transcriptome analysis of Staphylococcus aureus biofilms in response to innate immune cells |
|
