|
| Status |
Public on Nov 21, 2014 |
| Title |
B1917_5_mM.4 |
| Sample type |
RNA |
| |
|
| Source name |
ptxP1 strain, 5 mM sulfate
|
| Organism |
Bordetella pertussis B1917 |
| Characteristics |
strain: ptxP1
treatment: 5 mM sulfate
|
| Treatment protocol |
Bacteria were treated with 2 volumes of RNAprotect bacteria reagent (Qiagen).
|
| Growth protocol |
Cultures were grown at 37°C in chemically defined THIJS medium supplemented with 0.2 mg/ml Heptakis-cyclodextrin with different sulfate concentrations until a mid-log OD620 of 0.5 to 0.6 at which point the bacteria were harvested for RNA isolation .
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the RNeasy Mini kit (Qiagen) and DNA was removed by DNase digestion with the DNA-free kit (Ambion).
|
| Label |
Cy3
|
| Label protocol |
Total RNA (5 µg) was labeled according to standard Nimblegen gene expression array protocols.
|
| |
|
| Hybridization protocol |
2 µg of labeled cDNA was applied to 4x72K custom design NimbleGen arrays. Overnight hybridization at 42°C and subsequent washing of arrays was performed according to the manufacturer’s instructions (available from www.nimblegen.com).
|
| Scan protocol |
Array images were acquired with a NimbleGen MS200 scanner following their standard operation protocol, using the autogain function per subarray.
|
| Description |
ptxP1 strain, 5 mM sulfate
|
| Data processing |
Normalized expression data was analyzed by ArrayStar (DNASTAR, Madison, WI, USA) using the Robust Multiarray Analysis (RMA) algorithm for background correction and quantile normalization. For the identification of sulfate-modulated genes the raw expression data under low, medium, and high sulfate was normalized individually for each strain. To identify absolute differences in gene expression between the ptxP1 and ptxP3 strain, normalization was performed using the raw expression data of both strains under all sulfate conditions. Log2 transformed signals were used to generate kernel density plots using a Gaussian model with stepwise increasing bandwidth until a single local minimum was found between the distributions of background signal and gene expression. Positions where the first derivative of the density traverses from values below to values above zero were considered local minima. The corresponding expression value is the value closest to the minimum between the peaks of expressed and non-expressed genes and was therefore considered as a cutoff value to determine whether a gene was expressed or not.
|
| |
|
| Submission date |
Jul 31, 2013 |
| Last update date |
Nov 22, 2014 |
| Contact name |
Aldert Zomer |
| E-mail(s) |
[email protected]
|
| Organization name |
Utrecht University
|
| Department |
Department of Infectious Diseases and Immunology,Faculty of Veterinary Medicine
|
| Street address |
Yalelaan 1
|
| City |
Utrecht |
| ZIP/Postal code |
3584 cl |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL17516 |
| Series (1) |
| GSE49385 |
Differentially Expressed Genes in Bordetella pertussis Strains Belonging to a Lineage Which Recently Spread Globally |
|
