strain: 168 genotype: wild type treatment: methylglyoxal (MG)
Treatment protocol
At OD500 of 0.4, cells were treated for 10 minutes with 2.8 mM methylglyoxal.
Growth protocol
B. subtilis 168 wild type, delta ytpQ mutant, and delta spx mutant cells were grown under vigorous agitation at 37 °C in a minimal medium described before (Stülke et al., 1993, J Gen Microbiol 139, 2041-2045).
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated by the acid phenol method as described (Majumdar et al., 1991, Biotechniques 11: 94-101).
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).
At OD500 of 0.4, cells were treated for 10 minutes with 2.8 mM methylglyoxal.
Growth protocol
B. subtilis 168 wild type, delta ytpQ mutant, and delta spx mutant cells were grown under vigorous agitation at 37 °C in a minimal medium described before (Stülke et al., 1993, J Gen Microbiol 139, 2041-2045).
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated by the acid phenol method as described (Majumdar et al., 1991, Biotechniques 11: 94-101).
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
ytpQ_2_MG ytpQ mutant vs. wild type_MG treated_second biological replicate
Data processing
Data were extracted using the Agilent Feature Extraction software (version 10.5, Agilent Technologies).
