|
| Status |
Public on Feb 02, 2016 |
| Title |
L1_50B over L1_90A |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
L1_50B
|
| Organism |
Bacillus cereus ATCC 14579 |
| Characteristics |
strain: ATCC 14579
treatment: heat-treated spores 50 min after mixing with BHI
|
| Treatment protocol |
Cells were harvested at above mentioned timepoints (see overall design) by spinning down, resuspended in 1 ml TRI reagent (Ambion) and snap frozen in liquid nitrogen.
|
| Growth protocol |
Untreated and heat-treated (1 min 95C) B. cereus ATCC14579 spores were mixed with BHI and incubated with aeration (200rpm) at 30°C untill harvesting time (see overal design).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from samples in TRI-reagent by defrosting on ice, spores were mechanically disrupted by extensive bead beating in the presence of Lysing Matrix B beads. Direct-zol RNA MiniPrep was used according to the manufacturer’s instructions for on column RNA purification. Residual chromosomal DNA was removed by 30 min off column treatment of the samples with TURBO DNA-free Kit. The resulting mRNA was subsequently cleaned with RNeasy Mini Kit. The RNA quantity and quality were checked by UV spectroscopy and RNA 6000 Nanochip.
|
| Label |
cy5
|
| Label protocol |
Cy3 and Cy5 labeling of the cDNA was performed with CyScribe Post-Labeling kit (GE Healthcare)
|
| |
|
| Channel 2 |
| Source name |
L1_90A
|
| Organism |
Bacillus cereus ATCC 14579 |
| Characteristics |
strain: ATCC 14579
treatment: heat-treated spores 90 min after mixing with BHI
|
| Treatment protocol |
Cells were harvested at above mentioned timepoints (see overall design) by spinning down, resuspended in 1 ml TRI reagent (Ambion) and snap frozen in liquid nitrogen.
|
| Growth protocol |
Untreated and heat-treated (1 min 95C) B. cereus ATCC14579 spores were mixed with BHI and incubated with aeration (200rpm) at 30°C untill harvesting time (see overal design).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from samples in TRI-reagent by defrosting on ice, spores were mechanically disrupted by extensive bead beating in the presence of Lysing Matrix B beads. Direct-zol RNA MiniPrep was used according to the manufacturer’s instructions for on column RNA purification. Residual chromosomal DNA was removed by 30 min off column treatment of the samples with TURBO DNA-free Kit. The resulting mRNA was subsequently cleaned with RNeasy Mini Kit. The RNA quantity and quality were checked by UV spectroscopy and RNA 6000 Nanochip.
|
| Label |
cy3
|
| Label protocol |
Cy3 and Cy5 labeling of the cDNA was performed with CyScribe Post-Labeling kit (GE Healthcare)
|
| |
|
| |
| Hybridization protocol |
Hybridization was performed as described in van Melis et al., 2011
|
| Scan protocol |
Slides were scanned with Agilent G2505C scanner
|
| Description |
Expression ratio of L1_50B and L1_90A
|
| Data processing |
Microarrays were normalized using the approach reported previously (by van Melis et al 2011) for germinating spores involving the creation of so called synthetic microarrays. The background-corrected, raw signals (Cy3 and Cy5 channel) of all arrays were hierarchically clustered (Pearson correlation, complete linkage) using Genemaths XT. New synthetic arrays were defined from sample pairs showing the highest similarity in the clustering. The synthetic microarrays were Lowess normalized and all pairwise ratios were determined. The Lowess-normalized data are available on the series record in the file: cybert_ratios.xlsx. The sample data table contains the normalized Cy5/Cy3 values that were used in the first step of the analysis.
|
| |
|
| Submission date |
Sep 15, 2015 |
| Last update date |
Feb 02, 2016 |
| Contact name |
Jos Boekhorst |
| E-mail(s) |
Jos.Boekhorst@nizo.com
|
| Organization name |
NIZO food research
|
| Street address |
Kernhemseweg 2
|
| City |
Ede |
| ZIP/Postal code |
6718 ZB |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL19768 |
| Series (1) |
| GSE73043 |
Identification of CdnL, a putative transcriptional regulator involved in repair and outgrowth of heat-damaged Bacillus cereus spores |
|
