Cultures were harvested by centrifugation (Beckman CoulterTM AvantiTM J-20XP, JA10 Rotor, 20 min, 6000 rpm, 4°C), after different incubation times (44, 62, 82, 96 and 240 h). The culture broth was collected in 500 ml tubes and swirled briefly in an ethanol/dry ice bath to rapidly cool down the cultures and freeze the RNA profile. Subsequently, the broth was centrifuged at 6000 rpm for 20 min at 4°C (Beckman CoulterTM AvantiTM J-20XP, JA10 Rotor). The pellets were re-suspended in 0.1 M Tris-HCl and then re-centrifuged to obtain cell pellets that were subsequently shock-frozen in liquid nitrogen and stored at -80°C
Growth protocol
Cells of R. baltica were grown in batch culture (500ml) with mineral medium. The cultures were incubated at 28°C on a rotary shaker
Extracted molecule
total RNA
Extraction protocol
The culture pellets were re-suspended in 0.1 M Tris-HCL and then re-centrifuged to cell pellets that were shock-frozen in liquid nitrogen and stored at -80°C. Total RNA was isolated using the proposed protocol of the TRI Reagent® Kit by Ambion (Austin, USA). cDNA synthesis was performed using the SuperScript direct cDNA labeling system kit by Invitrogen (Karlsruhe, DE) according to the manufacturer's instructions with random hexamers and unlabeled dCTP/dUTP.
Label
Alexa546
Label protocol
cDNA was directly labeled using the PlatinumBrightTM nucleic acid labeling kit based on KREATECH´s patented Universal Linkage System (ULS) (Biocat, Heidelberg, Germany) according to the manufacturer's protocol
Cultures were harvested by centrifugation (Beckman CoulterTM AvantiTM J-20XP, JA10 Rotor, 20 min, 6000 rpm, 4°C), after different incubation times (44, 62, 82, 96 and 240 h). The culture broth was collected in 500 ml tubes and swirled briefly in an ethanol/dry ice bath to rapidly cool down the cultures and freeze the RNA profile. Subsequently, the broth was centrifuged at 6000 rpm for 20 min at 4°C (Beckman CoulterTM AvantiTM J-20XP, JA10 Rotor). The pellets were re-suspended in 0.1 M Tris-HCl and then re-centrifuged to obtain cell pellets that were subsequently shock-frozen in liquid nitrogen and stored at -80°C
Growth protocol
Cells of R. baltica were grown in batch culture (500ml) with mineral medium. The cultures were incubated at 28°C on a rotary shaker
Extracted molecule
total RNA
Extraction protocol
The culture pellets were re-suspended in 0.1 M Tris-HCL and then re-centrifuged to cell pellets that were shock-frozen in liquid nitrogen and stored at -80°C. Total RNA was isolated using the proposed protocol of the TRI Reagent® Kit by Ambion (Austin, USA). cDNA synthesis was performed using the SuperScript direct cDNA labeling system kit by Invitrogen (Karlsruhe, DE) according to the manufacturer's instructions with random hexamers and unlabeled dCTP/dUTP.
Label
Alexa647
Label protocol
cDNA was directly labeled using the PlatinumBrightTM nucleic acid labeling kit based on KREATECH´s patented Universal Linkage System (ULS) (Biocat, Heidelberg, Germany) according to the manufacturer's protocol
Hybridization protocol
Blocking, denaturing, hybridization, washing and drying of the slides with N2 were carried out in an automated hybridization station HS400 (Tecan, Crailsheim, Germany). The spotted arrays were blocked in prehybridization solution containing 250 mM NaCl, 5 mM Tris/HCl at pH 8.0, 50% formamide, 0.5x SSC, 0.05% BSA, and 1% blocking reagent from Roche Diagnostics, Mannheim, Germany for 45 min at 52°C. For hybridisation at least 2 μg of Alexa 546 dye-labeled and 2 μg of Alexa 647 dye-labeled total cDNA were combined and taken up in a final volume of 100 µl DIG Easy Hyb hybridization solution (Roche Diagnostics, Mannheim, Germany). After the blocking step the sample solution was applied to the arrays, denaturized at 95°C for 3 min and hybridized under stringent conditions at 52°C for over 12 hours. After hybridization slides were washed at RT in ULTRArray Low Stringency Wash Buffer (Ambion, Austin, USA) and dried by N2.
Scan protocol
Slides were scanned at a resolution of 5 μm using a ScanArray Express Microarray scanner (Perkin Elmer, Wellesley, USA) with varied laser power and sensitivity level of the photomultiplier tube (PMT) for each slide. The provided image analysis software ScanArray Express Version 4.0 was used for automatic spot detection and signal quantification of both fluorophores.
Description
Replicate_a
Data processing
Raw data were automatically processed using the in-house developed microarray data analysis software tool MADA (www.megx.net/mada). First of all the spot intensities were local background corrected (spot intensity minus spot background intensity). Then signals were only assessed as positive if mean spot pixel intensity was higher than the mean local background intensity plus 2 times the standard deviation of the mean local background pixel intensity. Each gene is spotted in 3 replicates. Spot replicates with poor quality were removed from the data set according to the outlier test of MADA. For this test, first the standard deviation of all replicates is computed. Second, one replicate is omitted and the standard deviation is recalculated, if the deviation differs more than 50% from the previous deviation, the omitted replicate is regarded as outlier. This procedure is alternately repeated for all replicates The expression is described through the ratio and intensity, where R is the fluorescence log ratio of the experiment time point relative to the control condition (e.g. R = log2 (result of channel 44 h / result of channel 62 h)) and I is the log mean fluorescence intensity (e.g. I = log10 (result of channel 44 h * result of channel 62 h)). Each data point represents a regulation factor (ratio) in a logarithmic scale for one gene calculated from the positive replicates for a particular probe coming from two RNA pools. Normalization was carried out by LOWESS fitting on an R-versus-I plot with a smoothing factor of 0.05. Each time point of the growth curve experiment was hybridized. The expression data (ratio) of more than one hybridization were combined to one expression data point (ratio) by average and the standard deviation was calculated. Differentially expressed genes are determinate by a fixed threshold cut off method (i.e. a two-fold increase or decrease) based on the self-self hybridization.
