growth condition: NH4 cultured treatment: Ni deplete
Growth protocol
Exponential growth in synthetic ocean water for the nitrogen source indicated and either total Nickel=5nM (Ni deplete) or total Ni =50 nM (Ni replete)
Extracted molecule
total RNA
Extraction protocol
Modified trizol protocol (lysis at 60oC for 1 hour) followed by Qiagen Rneasy with Dnase treatment
Label
Cy3
Label protocol
ix µg of Random Hexamer Primers (Invitrogen, Carlsbad, CA) were annealed to 2 µg of RNA in a total volume of 18.5 µl by heating the reaction to 70°C for 10 min, followed by quick chilling on ice. To this reaction was added 6 µl of 5× Reverse Transcriptase cDNA reaction buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl2), 3 µl 0.1 M DTT (Invitrogen), and 0.6 µl of a dNTP solution containing (25 mM each dATP, dGTP, and dCTP, 8 mM dTTP (Invitrogen) and 17 mM amino allyl-dUTP (Sigma)). Reverse Transcriptase Superscript II (Invitrogen) (400 U in 2 µl) was added and reactions were then incubated at 42°C for three hours to overnight. The RNA template was hydrolysed by adding 10 µl 1 M NaOH and 10 µl 0.5 M EDTA and heating to 65°C for 15 min. The solution was neutralized with 25 µl 1 M Tris pH 7.0 and cDNA was purified over QIAquick PCR purification columns replacing the Tris buffers with phosphate buffers (5 mM KPO4 pH 8, 80% EtOH) and (4 mM KPO4 pH 8) for column washing and cDNA elution respectively. Reactions were dried to completion in a Speed-Vac centrifuge. Aminoallyl-labelled cDNA was resuspended in 4.5 µl 0.1 M Na2HCO3, pH 9.3, plus 4.5 µl containing 63 µg of the appropriate ester dye (Cy3 or Cy5). The coupling reaction proceeded for 2 h at room temperature in the dark. Thirty-five µl 100 mM NaOAc, pH 5.2 was added to coupling reactions which were then purified with QIAquick PCR purification columns, using the supplied buffers. The cDNA probes, Cy3 and Cy5, were combined and dried to completion.
growth condition: NH4 cultured treatment: Ni replete
Growth protocol
Exponential growth in synthetic ocean water for the nitrogen source indicated and either total Nickel=5nM (Ni deplete) or total Ni =50 nM (Ni replete)
Extracted molecule
total RNA
Extraction protocol
Modified trizol protocol (lysis at 60oC for 1 hour) followed by Qiagen Rneasy with Dnase treatment
Label
Cy5
Label protocol
ix µg of Random Hexamer Primers (Invitrogen, Carlsbad, CA) were annealed to 2 µg of RNA in a total volume of 18.5 µl by heating the reaction to 70°C for 10 min, followed by quick chilling on ice. To this reaction was added 6 µl of 5× Reverse Transcriptase cDNA reaction buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl2), 3 µl 0.1 M DTT (Invitrogen), and 0.6 µl of a dNTP solution containing (25 mM each dATP, dGTP, and dCTP, 8 mM dTTP (Invitrogen) and 17 mM amino allyl-dUTP (Sigma)). Reverse Transcriptase Superscript II (Invitrogen) (400 U in 2 µl) was added and reactions were then incubated at 42°C for three hours to overnight. The RNA template was hydrolysed by adding 10 µl 1 M NaOH and 10 µl 0.5 M EDTA and heating to 65°C for 15 min. The solution was neutralized with 25 µl 1 M Tris pH 7.0 and cDNA was purified over QIAquick PCR purification columns replacing the Tris buffers with phosphate buffers (5 mM KPO4 pH 8, 80% EtOH) and (4 mM KPO4 pH 8) for column washing and cDNA elution respectively. Reactions were dried to completion in a Speed-Vac centrifuge. Aminoallyl-labelled cDNA was resuspended in 4.5 µl 0.1 M Na2HCO3, pH 9.3, plus 4.5 µl containing 63 µg of the appropriate ester dye (Cy3 or Cy5). The coupling reaction proceeded for 2 h at room temperature in the dark. Thirty-five µl 100 mM NaOAc, pH 5.2 was added to coupling reactions which were then purified with QIAquick PCR purification columns, using the supplied buffers. The cDNA probes, Cy3 and Cy5, were combined and dried to completion.
Hybridization protocol
Slides were pretreated for 2 h at 42°C in a 50-ml solution of 5 × SSC, 0.1% SDS and 1% BSA followed by four washes in MilliQ water, and three washes in isopropanol. Residual isopropanol was removed by brief centrifugation at 2500 g. Dried probes were resupended in 30 µl hybridization buffer (50% formamide, 5× SSC, 0.1% SDS and 100 µg ml−1 salmon sperm DNA). This mixture was heated for 10 min at 95°C and then applied to a pretreated slide under a cover slip. Hybridizations were carried out at 42°C for 16 h in a sealed hybridization chamber (Corning ♯2551), which was humidified with 20 µl of 5× SSC. Microarrays were washed once in ∼ 200 ml 2× SSC/0.1% SDS at 55°C for 10 min, once in 0.1× SSC/0.1% SDS for 10 min at room temperature, three times in 0.1× SSC for 2 min each, and finally in MilliQ water. They were scanned immediately or following storage in a dessicator in the dark.
Scan protocol
Slides were promptly scanned at a 10 μm resolution using an Axon 4000B scanner with GenePix 4.0 software (Molecular Devices, Sunnyvale, CA). Processing of the TIFF images from hybridized arrays was performed using TIGR-Spotfinder and TIGR-MIDAS (http://www.tm4.org).
Data processing
Statistical analysis was performed on log2-transformed signal ratios of the replicate spots using the Significance Analysis of Microarray (SAM) software (http://www-stat.stanford.edu/~tibs/SAM/) (Tusher et al., 2001).
