|
| Status |
Public on Jul 18, 2017 |
| Title |
Y. pestis, Kim53∆nlpD 37°C vs.Kim53 28°C |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
grown at 37°C
|
| Organism |
Yersinia pestis |
| Characteristics |
strain: Kim53 ∆nlpD
|
| Growth protocol |
For bacterial total RNA preparation, bacterial colonies were grown on BHIA plats for 48 h at 28°C , harvested and diluted in heart infusion broth (HIB) (BD, USA) supplemented with 0.2% xylose and 2.5 mM CaCl2 (Sigma-Aldrich, Israel) to an OD660 of 0.01 and grown over night (o.n.) at 28°C in a shaker (200 rpm). The resulting cultures were diluted in fresh broth to an OD660 of 0.05 and allowed to grow for 5 h at 37°C. Aliquots of ~5×108 cfu were collected by centrifugation and cells were immediately frozen at -70°C until use.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA were extracted using RNeasy Mini kit (QIAGEN) according to the manufacturer’s instructions. Residual DNA was removed by 15 min on-column digestion using the RNase-free DNase kit (QIAGEN).
|
| Label |
Cy3
|
| Label protocol |
Complementary RNA (cRNA) was produced from the total RNA samples and fluorescently labeled using the MessageAmpTM II-Bacteria kit (Ambion) according to the manufacturer’s instructions. Each experiment comprised of samples representing 4 different ciprofloxacin concentrations labeled with either Cy3-CTP or Cy5-CTP, which were co-hybridized with the growth control sample labeled with the 2nd dye (Cy5-CTP or Cy3-CTP, accordingly).
|
| |
|
| Channel 2 |
| Source name |
grown at 28°C
|
| Organism |
Yersinia pestis |
| Characteristics |
strain: Kimberley53
|
| Growth protocol |
For bacterial total RNA preparation, bacterial colonies were grown on BHIA plats for 48 h at 28°C , harvested and diluted in heart infusion broth (HIB) (BD, USA) supplemented with 0.2% xylose and 2.5 mM CaCl2 (Sigma-Aldrich, Israel) to an OD660 of 0.01 and grown over night (o.n.) at 28°C in a shaker (200 rpm). The resulting cultures were diluted in fresh broth to an OD660 of 0.05 and allowed to grow for 5 h at 37°C. Aliquots of ~5×108 cfu were collected by centrifugation and cells were immediately frozen at -70°C until use.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA were extracted using RNeasy Mini kit (QIAGEN) according to the manufacturer’s instructions. Residual DNA was removed by 15 min on-column digestion using the RNase-free DNase kit (QIAGEN).
|
| Label |
Cy5
|
| Label protocol |
Complementary RNA (cRNA) was produced from the total RNA samples and fluorescently labeled using the MessageAmpTM II-Bacteria kit (Ambion) according to the manufacturer’s instructions. Each experiment comprised of samples representing 4 different ciprofloxacin concentrations labeled with either Cy3-CTP or Cy5-CTP, which were co-hybridized with the growth control sample labeled with the 2nd dye (Cy5-CTP or Cy3-CTP, accordingly).
|
| |
|
| |
| Hybridization protocol |
A slide (containing 8 arrays) was hybridized with samples originating from 2 independent biological experiments representing the same time point, performed with Cy3-Cy5 dye swap. Each array represent hybridization of treated sample with its growth-control sample. Hybridization was done using the Agilent Gene Expression Hybridization Kit according to Agilent protocol for two-color Microarray-Based Gene Expression Analysis.
|
| Scan protocol |
Scanned on an Agilent DNA microarray G2505B scanner.
Images were quantified using Agilent Feature Extraction (FE) Software (version 9.5.1.1), with linear and LOWESS normalization.
|
| Data processing |
Agilent FE Software (9.5.1.1) was used for background subtraction and LOWESS normalization. Statistical analysis was performed using the Limma (Linear Models for Microarray Data) package from the Bioconductor project (http://www.bioconductor.org). The processed signal from the FE was read into Limma using the “read.maimages” function. Background subtraction and LOWESS normalization were performed for each array. Quantile normalization was applied between arrays. The Benjamini-Hochberg false discovery rate (FDR) was used to correct for multiple comparisons.
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| |
|
| Submission date |
Jul 17, 2017 |
| Last update date |
Jan 23, 2018 |
| Contact name |
Avital Tidhar |
| E-mail(s) |
avitalt@iibr.gov.il
|
| Phone |
972-8-9381509
|
| Organization name |
Israel Institute for Biological Research
|
| Department |
Biochemistry and molecular Genetics
|
| Street address |
P.O.B 19
|
| City |
Ness-Ziona |
| ZIP/Postal code |
7410001 |
| Country |
Israel |
| |
|
| Platform ID |
GPL21664 |
| Series (1) |
| GSE101490 |
The Yersinia pestis NlpD Lipoprotein is important for iron assimilation and is functionally linked to the twin-arginine translocation system |
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