strains: USA300 FPR3757 growth phase: control logarithmic growth and transient phase; linezolid stress transient and stanionary phase
Treatment protocol
An overnight culture was used to inoculate 100 ml medium to an OD540 of 0.02. Control samples were taken during logarithmic growth phase (OD500=0.9) and transient growth phase (OD540=3.5). Logarithmic growing cells (OD540=0.5) were stressed with 5 mg Linezolid per ml. Stress samples were taken one hour after stress (OD540=0.9), at the onset of the temprorary growth arrest (OD540=1.8) and after the cells reentered the growth cycle (OD540=2.2)
Growth protocol
S. aureus was cultured in LB at 37° and 120 rpm.
Extracted molecule
total RNA
Extraction protocol
Total RNA was prepared by acid-phenol extraction after mechanical cell disruption as described previously (Otto et al., 2010, Nat Commun).
Label
Cy3
Label protocol
Total RNA was prepared from three independent experiments. Synthesis and purification of fluorescently labeled cDNA were carried out according to Charbonnier et al. (2005) with minor modifications. In detail, 10 µg of total RNA were mixed with random primers (Promega, Madison, WI, USA) and spike-ins (Two-Color RNA Spike-In Kit, Agilent Technologies, Santa Clara, CA, USA). 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, Karlsruhe, Germany), 5 µl of 0.1 M DTT (Invitrogen, Karlsruhe, Germany), 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, Little Chalfont, UK) and 2 µl of SuperScript II reverse transcriptase (Invitrogen, Karlsruhe, Germany). 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, Karlsruhe, Germany) at room temperature for 30 min. Labeled cDNA was then purified using the CyScribe GFX Purification Kit (GE Healthcare, Little Chalfont, UK). The individual samples were labeled with Cy5, whereas the reference pool was labeled with Cy3.
strain: USA300 FPR3757 growth phase: stress second transient growth phase
Treatment protocol
An overnight culture was used to inoculate 100 ml medium to an OD540 of 0.02. Control samples were taken during logarithmic growth phase (OD500=0.9) and transient growth phase (OD540=3.5). Logarithmic growing cells (OD540=0.5) were stressed with 5 mg Linezolid per ml. Stress samples were taken one hour after stress (OD540=0.9), at the onset of the temprorary growth arrest (OD540=1.8) and after the cells reentered the growth cycle (OD540=2.2)
Growth protocol
S. aureus was cultured in LB at 37° and 120 rpm.
Extracted molecule
total RNA
Extraction protocol
Total RNA was prepared by acid-phenol extraction after mechanical cell disruption as described previously (Otto et al., 2010, Nat Commun).
Label
Cy5
Label protocol
Total RNA was prepared from three independent experiments. Synthesis and purification of fluorescently labeled cDNA were carried out according to Charbonnier et al. (2005) with minor modifications. In detail, 10 µg of total RNA were mixed with random primers (Promega, Madison, WI, USA) and spike-ins (Two-Color RNA Spike-In Kit, Agilent Technologies, Santa Clara, CA, USA). 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, Karlsruhe, Germany), 5 µl of 0.1 M DTT (Invitrogen, Karlsruhe, Germany), 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, Little Chalfont, UK) and 2 µl of SuperScript II reverse transcriptase (Invitrogen, Karlsruhe, Germany). 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, Karlsruhe, Germany) at room temperature for 30 min. Labeled cDNA was then purified using the CyScribe GFX Purification Kit (GE Healthcare, Little Chalfont, UK). The individual samples were labeled with Cy5, whereas the reference pool was labeled with Cy3.
Hybridization protocol
300 ng of Cy5-labeled cDNA and 300 ng of Cy3-labeled cDNA were hybridized together to the microarray following Agilent's hybridization, washing, and scanning protocol (Two-Color Microarray-Based Gene Expression Analysis, version 5.5)
Scan protocol
Intensity values were extracted and processed (background subtraction and LOWESS normalization) by means of the Feature Extraction software (version 10.5, Agilent Technologies, Santa Clara, CA, USA).
Description
V_3
Data processing
Data were extracted using the Agilent Feature Extraction software (version 10.5, Agilent Technologies).
