|
| Status |
Public on Oct 01, 2018 |
| Title |
Stationary phase III (max. OD + 10h)_1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Clostridioides difficile 630∆erm at stationary growth phase III (10 hours after max. OD)
|
| Organism |
Clostridioides difficile 630 |
| Characteristics |
growth phase: Stationary phase III (maximal OD + 10 h)
|
| Treatment protocol |
Samples were taken at different time points along the growth curve: exponential growth (exp), transient phase (trans), ODmax (stat1), ODmax + 5 h (stat2) and + ODmax 10 h (stat3).
|
| Growth protocol |
The C. difficile 630∆erm strain (DSM28645) (Hussain, HA, 2005) obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) was grown anaerobically at 37 °C in a defined medium containing 10 g/L casamino acids and 2 g/L glucose (Cartman and Minton, 2010; Neumann-Schaal et al. 2015).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Bacterial RNA was extracted using the Qiagen RNeasy Kit Mini (Qiagen, Hilden, Germany) according to manufacturer’s instructions with minor modifications. Briefly, pellets from a 45 mL culture were resolved in 200 µl TE buffer and 15 mg/ml lysozyme, incubated for 30 min at room temperature and vigorously mixed every 2 min for 10 sec for enzymatically disruption of cells. Approximately 700 µL of RLT buffer and one spatula of glass beads were added and mixed vigorously for 3 min for mechanical disruption of cells. Samples were centrifuged (3 min, 10.000 rpm) and supernatant was mixed with 470 µL of 100% ethanol. Afterwards, RNA purification was carried out using the Qiagen RNeasy Kit protocol. DNA contaminating the RNA samples was removed using RNase-free DNase I twice (Qiagen, Hilden, Germany). The RNA was further assessed by analysis on the Bionanalyzer 2100 (Agilent, Santa Clara, CA) and RNA 6000 Nano Reagents (Agilent, Santa Clara, CA). According to the amount of RNA extracted and its quality (RINs>8), the best samples per time point were used for transcriptomic analysis.
|
| Label |
cy3
|
| Label protocol |
RNA samples were labelled with Cy3 (later growth phases) and Cy5 (exponential growth phase) using the ULS fluorescent labelling kit (Kreatech, Germany).
|
| |
|
| Channel 2 |
| Source name |
Clostridioides difficile 630∆erm at exponantial growth phase
|
| Organism |
Clostridioides difficile 630 |
| Characteristics |
growth phase: exponantial growth phase
|
| Treatment protocol |
Samples were taken at different time points along the growth curve: exponential growth (exp), transient phase (trans), ODmax (stat1), ODmax + 5 h (stat2) and + ODmax 10 h (stat3).
|
| Growth protocol |
The C. difficile 630∆erm strain (DSM28645) (Hussain, HA, 2005) obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) was grown anaerobically at 37 °C in a defined medium containing 10 g/L casamino acids and 2 g/L glucose (Cartman and Minton, 2010; Neumann-Schaal et al. 2015).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Bacterial RNA was extracted using the Qiagen RNeasy Kit Mini (Qiagen, Hilden, Germany) according to manufacturer’s instructions with minor modifications. Briefly, pellets from a 45 mL culture were resolved in 200 µl TE buffer and 15 mg/ml lysozyme, incubated for 30 min at room temperature and vigorously mixed every 2 min for 10 sec for enzymatically disruption of cells. Approximately 700 µL of RLT buffer and one spatula of glass beads were added and mixed vigorously for 3 min for mechanical disruption of cells. Samples were centrifuged (3 min, 10.000 rpm) and supernatant was mixed with 470 µL of 100% ethanol. Afterwards, RNA purification was carried out using the Qiagen RNeasy Kit protocol. DNA contaminating the RNA samples was removed using RNase-free DNase I twice (Qiagen, Hilden, Germany). The RNA was further assessed by analysis on the Bionanalyzer 2100 (Agilent, Santa Clara, CA) and RNA 6000 Nano Reagents (Agilent, Santa Clara, CA). According to the amount of RNA extracted and its quality (RINs>8), the best samples per time point were used for transcriptomic analysis.
|
| Label |
cy5
|
| Label protocol |
RNA samples were labelled with Cy3 (later growth phases) and Cy5 (exponential growth phase) using the ULS fluorescent labelling kit (Kreatech, Germany).
|
| |
|
| |
| Hybridization protocol |
300 ng of Cy3-labeled RNA and 300 ng of Cy5-labeled RNA were mixed and fragmented, and hybridized to the microarray at 65 °C for 17 hours using the Agilent Hybridization Chamber according to the manufacturer’s instructions.
|
| Scan protocol |
The microarrays were scanned with the help of the Agilent C Scanner (Agilent Technologies, USA) using the software Agilent Scan Controll 8.4.1
|
| Data processing |
For feature extraction the software Feature Extraction 10.7.3.1 (Agilent Technologies, USA) was used, data processing was carried out using the Bioconducter – Linear Models for Microarray analysis (LIMMA) package of the R language (http://www.r-project.org).
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| |
|
| Submission date |
May 29, 2018 |
| Last update date |
Sep 27, 2019 |
| Contact name |
Rebekka Biedendieck |
| Organization name |
Technische Universtität Braunschweig
|
| Department |
Institute of Microbiology
|
| Street address |
Rebenring 56
|
| City |
Braunschweig |
| ZIP/Postal code |
38106 |
| Country |
Germany |
| |
|
| Platform ID |
GPL25054 |
| Series (1) |
| GSE115054 |
Metabolic reprogramming with the induction of toxin production of Clostridioides difficile during the stationary phase |
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