Mean foreground values were used for the analysis. Slides were normalized and filtered using the LIMMA on the R 2.7.1 platform was used. Spots with negative flags from GenePix, and spots with two low channels or two saturated channels were all removed, controls were weighed down, remaining spots were weighted according to quality. An offset was added to the data before normalisation with global loess. After normalisation, controls were removed and replicates within each slide were averaged. As the positive controls were often found to be significant, these were normalized and averaged separately.
fter normalisation, controls were removed and replicates within each slide were averaged. The averaged values were compared across slides and a linear model was fitted to the data for each gene. The model was evaluated by Bayesian statistics (9-10), and moderated t-statistics were computed. On basis of this, p-values were computed using false discovery rate correction (fdr). The script used for data analysis can be found in file nprR_script_for_AE.txt, which has been included in this submission.
The slides were scanned with an Axon 4000B scanner. Intensities for the red and green channels were adjusted to give a ratio close to 1 between the signals in the two channels for the slide as a whole. Gridding, spot annotation and calculation of raw spot intensities was done with the GenePix Pro 6.1 software.
The microarray slides were prehybridized before use for 30-60 minutes in a 5xSSC/0.1 % SDS/0.1 % BSA solution at 42 degrees C, according to the UltraGAPS(TM) Coated Slides instruction manual from Corning. The slides were then washed three times in MQ H2O, once in isopropanol and finally spun dry. 45 uL hybridization solution was added to the samples to a final concentration of 30 % formamide, 5xSSC, 0.1 % SDS and 0.1 mg/mL sperm DNA, based on the UltraGAPS(TM) Coated Slides instruction manual from Corning (USA). Labeled cDNA were denatured at 95 degrees C for 2 minutes, and incubated at 42 degrees C before hybridization. The samples were hybridized in a hybridization chamber (Monterey Industries, CA, USA), humidified with 5xSSC for 16 hours in a 42 degrees C water bath. After hybridization, the slides were washed at 42 degrees C in 0.5xSSC/0.01 % SDS and in 0.06xSSC, and finally at room temperature in isopropanol before they were spun dry.
1 and 3 hours after the break in the logarithmic growth curve (After 3.5 and 5.5 hours growth respectively), samples were harvested directly into an equal volume of ice cold methanol, incubated at room temperature for 5 minutes, and centrifuged at 3800 g and 4 degrees C. The supernatant was removed and the pellets resuspended in 1 mL 15 mg/mL lysozyme. The suspension was incubated at room temperature for 20 minutes and frozen at -70 degrees C after addition of RLT buffer from the RNeasyﾙ midi kit (Qiagen, Germany).
cDNA synthesis, labelling and purification was done with the FairPlay(TM) microarray labeling kit (Stratagene, CA, USA), using 500 ng random hexamers (Applied Biosystems, CA, USA) on 20 ug of each RNA, and with amino-allyl coupling of Cy3 and Cy5 dyes from Amersham Biosciences (GE Healthcare Bio-Sciences AB, Sweden). After purification, the samples were concentrated with a microcon column (Millipore, MA, USA). Final volume was adjusted to 35 uL.
Samples were thawed for 15 minutes at 37 degrees C. RNA isolation, including DNase treatment, was performed with the RNeasy Midi Kit (Qiagen, Germany) together with the RNase-Free DNase Set (Qiagen, Germany).
Single colonies from fresh plates of Bacillus thuringiensis 407 nprA::lacZ and Bacillus thuringiensis 407 nprA::lacZ delta-nprR -nprX were used to inoculate 10 mL of LB, which was incubated for 17 hours at 30 degrees C and 250 rpm. 1 L baffled erlenmeyerflasks with 100 mL LB were inoculated with the overnight culture and incubated at 37 degrees C and 250 rpm.