The common reference was created by pooling 400 ng Cy5-labeled RNA sample of each time point of each growth curve, 35 points in total.
Growth protocol
MSSA476 was grown overnight in Iscove’s Modified Dulbecco’s Medium (IMDM) (Invitrogen, Carlsbad, CA, USA). These overnight cultures were diluted in fresh prewarmed IMDM and grown twice to mid-log phase culture (A660 ~0.5) prior to the growth experiment. The second midlog phase culture was diluted to an A660 of 0.3 with prewarmed IMDM and directly transferred to fresh prewarmed IMDM to obtain an A660 of 0.03. Samples were taken at 1, 2, 3, 4, 5, 6 and 9 h post inoculation (p.i.). A660 was measured and dilutions were plated on sheep blood-agar plates to determine colony forming units (CFUs). Cultures were incubated at 37°C and 180 rpm.
Extracted molecule
total RNA
Extraction protocol
RNA extraction was performed at room temperature (RT) unless stated otherwise. RNA was purified using the NucleoSpin RNA II total RNA isolation kit (Macherey-Nagel, Düren, Germany) according to manufacturer’s protocol with some adjustments. Bacteria were spun 30 sec at 13000 rpm, immediately resuspended in 350 µl RA1 buffer supplemented with 3.5 µl β-mercaptoethanol (Sigma-Aldrich, Zwijndrecht, The Netherlands) and vortexed vigorously. Resuspended bacteria were added to 0.5 ml 0.1 mm silica beads (Merlin, Breda, The Netherlands), disrupted using a mini-Beadbeater (BioSpec Products, Bartlesville, OK, USA) for 30 sec at 5000 rpm and samples were frozen at -20°C overnight. The samples were thawed slowly and purified. Total RNA was eluted in 60 µl RNase free MilliQ. RNA yield was measured using a NanoDrop ND-1000 (Nanodrop Technologies, Wilmington, DE, USA) and quality was measured using a 2100 BioAnalyzer (Agilent Technologies, Palo Alto, CA, USA). Both the RNA integrity number (RIN) and the presence of degradation products were checked.
Label
Cy5
Label protocol
Total RNA was labeled in a one-step labeling with fluorescent dyes by direct labeling. A total of 10 µg RNA was randomly primed with Superscript II reverse transcriptase (Invitrogen) and random hexamers, in a total volume of 30 µl, for 2 h at 42°C with the incorporation of Cy5- or Cy3-dUTP (Agilent Technologies) with a ratio dUTP/dTTP of 3/1, yielding approximately 4 µg labeled cDNA. RNA template was removed by hydrolysis with 3 µl 2.5 M NaOH (Sigma-Aldrich) for 15 min at 70°C. Hydrolysis was stopped by neutralization with 15 µl 2 M MOPS (Sigma-Aldrich) and put on ice. Labeled cDNA was purified using Qia-quick PCR purification kit (Qiagen, Valencia, CA, USA). Incorporation of Cy3 or Cy5 was determined using a NanoDrop ND-1000.
MSSA476 was grown overnight in Iscove’s Modified Dulbecco’s Medium (IMDM) (Invitrogen, Carlsbad, CA, USA). These overnight cultures were diluted in fresh prewarmed IMDM and grown twice to mid-log phase culture (A660 ~0.5) prior to the growth experiment. The second midlog phase culture was diluted to an A660 of 0.3 with prewarmed IMDM and directly transferred to fresh prewarmed IMDM to obtain an A660 of 0.03. Samples were taken at 1, 2, 3, 4, 5, 6 and 9 h post inoculation (p.i.). A660 was measured and dilutions were plated on sheep blood-agar plates to determine colony forming units (CFUs). Cultures were incubated at 37°C and 180 rpm.
Extracted molecule
total RNA
Extraction protocol
RNA extraction was performed at room temperature (RT) unless stated otherwise. RNA was purified using the NucleoSpin RNA II total RNA isolation kit (Macherey-Nagel, Düren, Germany) according to manufacturer’s protocol with some adjustments. Bacteria were spun 30 sec at 13000 rpm, immediately resuspended in 350 µl RA1 buffer supplemented with 3.5 µl β-mercaptoethanol (Sigma-Aldrich, Zwijndrecht, The Netherlands) and vortexed vigorously. Resuspended bacteria were added to 0.5 ml 0.1 mm silica beads (Merlin, Breda, The Netherlands), disrupted using a mini-Beadbeater (BioSpec Products, Bartlesville, OK, USA) for 30 sec at 5000 rpm and samples were frozen at -20°C overnight. The samples were thawed slowly and purified. Total RNA was eluted in 60 µl RNase free MilliQ. RNA yield was measured using a NanoDrop ND-1000 (Nanodrop Technologies, Wilmington, DE, USA) and quality was measured using a 2100 BioAnalyzer (Agilent Technologies, Palo Alto, CA, USA). Both the RNA integrity number (RIN) and the presence of degradation products were checked.
Label
Cy3
Label protocol
Total RNA was labeled in a one-step labeling with fluorescent dyes by direct labeling. A total of 10 µg RNA was randomly primed with Superscript II reverse transcriptase (Invitrogen) and random hexamers, in a total volume of 30 µl, for 2 h at 42°C with the incorporation of Cy5- or Cy3-dUTP (Agilent Technologies) with a ratio dUTP/dTTP of 3/1, yielding approximately 4 µg labeled cDNA. RNA template was removed by hydrolysis with 3 µl 2.5 M NaOH (Sigma-Aldrich) for 15 min at 70°C. Hydrolysis was stopped by neutralization with 15 µl 2 M MOPS (Sigma-Aldrich) and put on ice. Labeled cDNA was purified using Qia-quick PCR purification kit (Qiagen, Valencia, CA, USA). Incorporation of Cy3 or Cy5 was determined using a NanoDrop ND-1000.
Hybridization protocol
Labeled cDNA was hybridized according to manufacturer’s protocol (Agilent Technologies). A total of 300 ng Cy3-labeled cDNA and 300 ng Cy5-labeled common reference was mixed, 10x Blocking agent was added to a total volume of 25 µl. The mixture was heated to 95°C for 3 min, followed by addition of 2x hybridization buffer to a volume of 50 µl. A total of 40 µl was loaded onto an 8x15k array and hybridized for 18 hours at 65°C and 20 rpm in a dedicated hybridization oven (Agilent Technologies). After the hybridization the arrays were washed in buffer 1 for 1 min at RT, then 5 min in wash buffer 1 at RT and finally 1 min in wash buffer 2 at 37°C (Agilent Technologies). Slides were spun for 3 min at 300 rpm to dry and scanned immediately.
Scan protocol
Data was extracted and processed using Feature Extractiontm software (version 9.5.1, Agilent Technologies). Median spot intensities were extracted by Feature Extraction software (version 9.5.1, Agilent Technologies).
Description
The common reference was created by pooling 400 ng Cy5-labeled RNA sample of each time point of each growth curve, 35 points in total.
Data processing
Processing of the data was performed using R (version 2.7.0) and the Bioconductor MAANOVA package (version 1.10.0). All slides were subjected to a set of quality control checks, i.e. visual inspection of the scans, examination of the consistency among the replicated samples by principal component analysis, testing against criteria for signal to noise ratios, testing for consistent performance of the labelling dyes and visual inspection of pre- and post-normalized data with box and ratio-intensity plots. After log2 transformation, the data were normalized by a LOWESS smoothing procedure to correct for dye bias effects.
