DNA manipulations were carried by standard techniques, chromosomal DNA was purified using a phenol chloroform extraction. Transformations of cells were carried out as described previously {Inoue, 1990 #1033;Anagnostopoulos, 1961 #1416;Hamoen, 2002 #1417}, and plated on nutrient agar supplemented with ampicillin (100 ug/ml), chloramphenicol (5 ug/ml), erythromycin (1 ug/ml), glucose (0.4 0.8%), kanamycin (5 ug/ml), spectinomycin (50 ug/ml), tetracycline (12 ug/ml), X gal (160 mg/ml), IPTG (0.5 1 mM), xylose (0.025 1 %).
Growth protocol
Bacterial cultures were grown at 30ºC or 37ºC in liquid LB medium, or on solid nutrient agar (Oxoid).
Extracted molecule
total RNA
Extraction protocol
Cell pellets were flash frozen in liquid nitrogen immediately after harvesting and stored at -80°C prior to RNA extraction. Frozen pellets were ground using a mortar and pestle before immersion in 300 μl Qiazol reagent (Qiagen). RNA was isolated using the manufacturer’s Qiazol protocol, with the exception that the RNA-containing water phase was further purified using the E.Z.N.A. MicroElute RNA Clean-up Kit (Omega Biotek), including an on-column treatment with the RNase-free DNase set (Life Technologies) to remove DNA contamination. The amount of RNA was measured on a NanoDrop ND-1000 (Thermo Scientific). RNA integrity was measured with a BioAnalyzer (Agilent Technolgies) using the RNA nano 6000 kit (Agilent Technologies) yielding RIN-values ≥ 9.7.
Label
Cy3
Label protocol
For each sample, 5 µg total RNA was combined with 1 µg random octamers (Biolegio) in a total volume of 4.5 µl, heat-denatured at 65°C for 10 min and immediately transferred to ice-water for 10 min. Subsequently, 10 µl of a first-strand mastermix was added containing final concentrations of 50 mM Tris-Cl (pH 8.3), 3 mM MgCl2, 75 mM KCl, 200 mM Raffinose (Sigma-Aldrich), 0.015% Triton X-100, 30 ng Actinomycin-D (Sigma-Aldrich), 0.01M DTT, 0.5 mM dGAC, 0.35 mM dUTP, 0.15 mM dUTP-Cy3 (test sample) or dUTP-Cy5 (common reference) (GE Healthcare) and 200U SuperScript-II (Life Technologies). This mixture was incubated for 2 min at 25°C, 120 min at 42°C and 15 min at 70°C. Finally, RNA was hydrolyzed by adding 1.5 µl 2.5 M NaOH and incubating for 10 min at 70°C. 8.5 µl 2M MOPS was added for neutralization and the labeled cDNA was purified with the E.Z.N.A. MicroElute RNA Clean-up Kit (Omega Biotek). Dye incorporation and cDNA yield was measured on the NanoDrop ND-1000 (Thermo Scientific) yielding 2-2.5 µg per sample and a FOI > 8 pmol/µg. The common reference was made by an equimolar pool of all test samples (5 µg per sample) and subsequently labeled with Cy5 as described above.
Hybridization protocol
Each hybridization mixture was made up from 1.1 µg test (Cy3) and 1.1 µg reference (Cy5) sample. Samples were dried and 1.98 µl of the appropriate sample tracking control (STC, Roche Nimblegen) was added. The hybridization cocktail was prepared according to the manufacturer’s instructions (Nimblegen Arrays User’s Guide – Gene Expression Arrays Version 5.0, Roche Nimblegen) and 5.22 µl was added to each sample. The samples were incubated for 5 min at 95°C and 5 min at 42°C prior to loading. Hybridization samples were loaded onto a XXXXXXXXXXX microarray (Design ………… EXP HX12). Microarrays were hybridized for 20 hours at 42°C with the NimbleGen Hybridization System 4 (Roche Nimblegen).
Scan protocol
Afterwards, the slides were washed according to the Nimblegen Arrays User’s Guide – Gene Expression Arrays Version 5.0 and scanned in an ozone-free room with a Agilent DNA microarray scanner G2565CA (Agilent Technologies).
Description
Bacillus subtilis KS696 gFG_527538A07
Data processing
Feature extraction was performed with NimbleScan v2.5 (Roche Nimblegen).
