wt salmonella mutant grown to 0d600 0.4 in 25 ml e salts minimal medium supplemented with 0.4% glucose, 0.1% casamino acids, 10μm fecl2, and 2 μg/ml thiamine, let grow for an additional 30 min, replicate 05: 110208
Extracted molecule
total RNA
Extraction protocol
High Pure RNA isolation kit (Roche)
Label
Cy5
Label protocol
Incorporation of dye using about 1200U Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA), 40ng/ul random hexamer primers, 4nmol Cy dUTP (GE Healthcare, Fairfield, CT), and 80U RNAse Inhibitor (Roche, Indianapolis, IN), in 60ul reactions. Reverse transcription was performed for 2h at 42˚C, and the pH adjusted with 50mM NaOH. After a 10min incubation at 70˚C reactions were neutralized using HCl. Labeled cDNA was purified using the QIAGEN PCR purification kit as suggested by the manufacturer (Qiagen, Valencia, CA).
wt salmonella mutant grown to 0d600 0.4 in 25 ml e salts minimal medium supplemented with 0.4% glucose, 0.1% casamino acids, 10μm fecl2, and 2 μg/ml thiamine, and treated with 5 mm detanonoate for 30 min, replicate 05: 110208
Extracted molecule
total RNA
Extraction protocol
High Pure RNA isolation kit (Roche)
Label
Cy3
Label protocol
Incorporation of dye using about 1200U Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA), 40ng/ul random hexamer primers, 4nmol Cy dUTP (GE Healthcare, Fairfield, CT), and 80U RNAse Inhibitor (Roche, Indianapolis, IN), in 60ul reactions. Reverse transcription was performed for 2h at 42˚C, and the pH adjusted with 50mM NaOH. After a 10min incubation at 70˚C reactions were neutralized using HCl. Labeled cDNA was purified using the QIAGEN PCR purification kit as suggested by the manufacturer (Qiagen, Valencia, CA).
Hybridization protocol
42C overnight in standard hybridization buffer (Roche Nimblegen)
Scan protocol
GenePix4000B scanner (Molecular Devices, Sunnyvale, CA) using Acuity 4.0 software.
Data processing
Signal extraction and quantitation with Roche’s NimbleScan 2.4 package
