Thermotoga species were grown at 80°C in an orbital shaking bath on artificial sea water media supplemented with either 2.5 g/L glucose or a polysaccharide mixture consisting of 0.12 g/L of each of the following: galactomannan, glucomannan, xylan, pectin, lichenan, and carboxymethyl cellulose. Cultures were sub-cultured at least six times with the appropriate carbon source in 50 mL cultures before being inoculated into 350 mL cultures for RNA extraction.
Extracted molecule
total RNA
Extraction protocol
At harvest, cultures were cooled in a dry ice-ethanol bath before centrifugation. Total RNA was extracted using TRIzol (Invitrogen) and purified using an RNeasy kit (Qiagen) with on-column DNase treatment.
Label
Cy3,Cy5
Label protocol
Superscript III (Invitrogen) was used to reverse transcribe RNA and incorporate 5-(3-aminoallyl)-dUTP (Ambion). cDNA was labeled with Cy5 or Cy3 dye (GE Healthcare).
treatment: galactomannan, glucomannan, xylan, pectin, lichenan, and carboxymethyl cellulose
Growth protocol
Thermotoga species were grown at 80°C in an orbital shaking bath on artificial sea water media supplemented with either 2.5 g/L glucose or a polysaccharide mixture consisting of 0.12 g/L of each of the following: galactomannan, glucomannan, xylan, pectin, lichenan, and carboxymethyl cellulose. Cultures were sub-cultured at least six times with the appropriate carbon source in 50 mL cultures before being inoculated into 350 mL cultures for RNA extraction.
Extracted molecule
total RNA
Extraction protocol
At harvest, cultures were cooled in a dry ice-ethanol bath before centrifugation. Total RNA was extracted using TRIzol (Invitrogen) and purified using an RNeasy kit (Qiagen) with on-column DNase treatment.
Label
Cy5,Cy3
Label protocol
Superscript III (Invitrogen) was used to reverse transcribe RNA and incorporate 5-(3-aminoallyl)-dUTP (Ambion). cDNA was labeled with Cy5 or Cy3 dye (GE Healthcare).
Hybridization protocol
Microarray slides were pre-hybridized at 42°C for 45 min in 5X SSC, 0.1% SDS, 0.1% BSA, dipped five times in sterile dH2O, dipped twice in isopropanol, and dried in a slide spinner. cDNA was concentrated, resuspended in sterile dH2O, and heated to 95°C for 2 min. 2X hybridization buffer (5X SSC, 5X Denhardt's solution, 30% (v/v) formamide, 0.1% SDS) was added to equal part cDNA. This mixture was applied to the slide and covered with a 20x60 mm polyethylene hydrophobic coverslip. The slide was placed in a hybridization chamber (Corning) and incubated at 42°C for 18-20 hours. Slides were washed for 4 min in buffer 1 (2X SSC, 0.2% SDS), 4 min in buffer 2 (0.5X SSC, 0.2% SDS), and 4 min in buffer 3 (0.5X SSC). Then slides were dried using a slide spinner.
Scan protocol
Slides for T. neapolitana and T. sp. RQ2 were scanned with an Perkin Elmer ScanArray Lite scanner and ScanArray 2.1 software, while T. maritima and T. petrophila slides were scanned with an Axon GenePix 4000b scanner and GenePix Pro 6.0 software.
Data processing
Spots were identified, quantified, and background intensity was subtracted using ScanArray 2.1 or GenePix Pro 6.0. Data processing was performed using JMP Genomics (SAS) and included log2 transformation, lowess normalization, ANOVA normalization, and row-by-row ANOVA modeling.
