|
| Status |
Public on Sep 01, 2011 |
| Title |
B. subtilis_RNaseY depletion_replicate A_hyb2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
B. subtilis GP193_without xylose
|
| Organism |
Bacillus subtilis subsp. subtilis str. 168 |
| Characteristics |
strain: GP193
treatment: no xylose
|
| Treatment protocol |
Cells were harvested immediately after the first detectable change in growth rate of the culture without xylose due to the reduced amounts of RNase Y.
|
| Growth protocol |
The B. subtilis strain GP193 expressing the essential gene rny (encoding RNase Y) under a xylose inducible promoter was grown under vigorous agitation at 37 °C in CSE minimal medium (Wacker et al., 2003, Microbiology 149: 3001-3009) in the presence and absence of xylose.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by the mechanical cell disruption/ acid phenol method (Eymann et al., 2002, J. Bacteriol. 184: 2500-2520).
|
| Label |
Cy3
|
| Label protocol |
For cDNA synthesis, 10 µg of total RNA were mixed with random primers (Promega) and spike-ins (Two-Color RNA Spike-In Kit, Agilent Technologies). The RNA/primer mixture was incubated at 70 °C for 10 min followed by 5 min incubation on ice. Then, the following reagents were added: 10 µl of 5x First Strand Buffer (Invitrogen), 5 µl of 0.1 M DTT (Invitrogen), 0.5 µl of a dNTP mix (10 mM dATP, dGTP, and dTTP, 2.5 mM dCTP), 1.25 µl of Cy3-dCTP or Cy5-dCTP (GE Healthcare) and 2µl of SuperScript II reverse transcriptase (Invitrogen). The reaction mixture was incubated at 42 °C for 60 min and then heated to 70 °C for 10 min. After 5 min on ice, the RNA was degraded by incubation with 2 units of RNaseH (Invitrogen) at room temperature for 30 min. Labeled cDNA was then purified using the CyScribe GFX Purification Kit (GE Healthcare).
|
| |
|
| Channel 2 |
| Source name |
B. subtilis GP193_with xylose
|
| Organism |
Bacillus subtilis subsp. subtilis str. 168 |
| Characteristics |
strain: GP193
treatment: with xylose
|
| Treatment protocol |
Cells were harvested immediately after the first detectable change in growth rate of the culture without xylose due to the reduced amounts of RNase Y.
|
| Growth protocol |
The B. subtilis strain GP193 expressing the essential gene rny (encoding RNase Y) under a xylose inducible promoter was grown under vigorous agitation at 37 °C in CSE minimal medium (Wacker et al., 2003, Microbiology 149: 3001-3009) in the presence and absence of xylose.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by the mechanical cell disruption/ acid phenol method (Eymann et al., 2002, J. Bacteriol. 184: 2500-2520).
|
| Label |
Cy5
|
| Label protocol |
For cDNA synthesis, 10 µg of total RNA were mixed with random primers (Promega) and spike-ins (Two-Color RNA Spike-In Kit, Agilent Technologies). The RNA/primer mixture was incubated at 70 °C for 10 min followed by 5 min incubation on ice. Then, the following reagents were added: 10 µl of 5x First Strand Buffer (Invitrogen), 5 µl of 0.1 M DTT (Invitrogen), 0.5 µl of a dNTP mix (10 mM dATP, dGTP, and dTTP, 2.5 mM dCTP), 1.25 µl of Cy3-dCTP or Cy5-dCTP (GE Healthcare) and 2µl of SuperScript II reverse transcriptase (Invitrogen). The reaction mixture was incubated at 42 °C for 60 min and then heated to 70 °C for 10 min. After 5 min on ice, the RNA was degraded by incubation with 2 units of RNaseH (Invitrogen) at room temperature for 30 min. Labeled cDNA was then purified using the CyScribe GFX Purification Kit (GE Healthcare).
|
| |
|
| |
| Hybridization protocol |
500 ng of Cy5-labeled cDNA and 500 ng of Cy3-labeled cDNA were hybridized to the microarray following Agilent’s hybridization, washing and scanning protocol (Two-Color Microarray-based Gene Expression Analysis, version 5.5).
|
| Scan protocol |
The microarray was scanned using an Agilent Microarray Scanner G2565CA (Agilent Technologies).
|
| Description |
B. subtilis_RNase Y depletion_first biological replicate_dye swap
|
| Data processing |
Data were extracted using the Agilent Feature Extraction software (version 10.5, Agilent Technologies).
|
| |
|
| Submission date |
Jul 06, 2011 |
| Last update date |
Sep 01, 2011 |
| Contact name |
Ulrike Mäder |
| E-mail(s) |
ulrike.maeder@uni-greifswald.de
|
| Organization name |
University Medicine Greifswald
|
| Department |
Functional Genomics
|
| Street address |
F.-L.-Jahn-Str. 15A
|
| City |
Greifswald |
| ZIP/Postal code |
D-17489 |
| Country |
Germany |
| |
|
| Platform ID |
GPL10901 |
| Series (1) |
| GSE30430 |
RNA processing in Bacillus subtilis: Identification of targets of the essential RNase Y |
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