Exponential phase culture of Caulobacter strain CB15N grown in M2-Glucose media.
Extracted molecule
total RNA
Extraction protocol
Cultures were spun down at 10,000 x g for 30 seconds, the supernatant was removed, and the cell pellets were flash frozen in liquid nitrogen. RNA was isolated from these cells by incubating in 1 ml of Trizol (Invitrogen, Carlsbad, CA) at 65°C for 10 minutes, adding chloroform, vortexing, spinning, and extracting the aqueous layer. Nucleic acid in the aqueous layer was isopropanol precipitated overnight at -80°C followed by a 30 minute centrifugation at 16,000 x g. The ethanol-washed and air-dried nucleic acid pellet was resuspended in 50 μl of nuclease-free water (IDT, Coralville, IA). 1 μl of RNase-free DNase I (Ambion, Austin, TX) was added to the sample and incubated at room temperature for two hours to remove any residual DNA. The nucleic acid in this digested sample was then acid phenol-chloroform (Ambion, Austin, TX) extracted, ethanol precipitated at -80°C overnight, and centrifuged at 16,000 x g to produce a DNA-free RNA pellet. RNA quality was assessed via agarose gel electrophoresis and RNA concentration determined by UV spectrophotometry using a Shimadzu UV-1650 (Kyoto, Japan).
Exponential phase culture of Caulobacter strain CB15N grown in M2-Inositol media.
Extracted molecule
total RNA
Extraction protocol
Cultures were spun down at 10,000 x g for 30 seconds, the supernatant was removed, and the cell pellets were flash frozen in liquid nitrogen. RNA was isolated from these cells by incubating in 1 ml of Trizol (Invitrogen, Carlsbad, CA) at 65°C for 10 minutes, adding chloroform, vortexing, spinning, and extracting the aqueous layer. Nucleic acid in the aqueous layer was isopropanol precipitated overnight at -80°C followed by a 30 minute centrifugation at 16,000 x g. The ethanol-washed and air-dried nucleic acid pellet was resuspended in 50 μl of nuclease-free water (IDT, Coralville, IA). 1 μl of RNase-free DNase I (Ambion, Austin, TX) was added to the sample and incubated at room temperature for two hours to remove any residual DNA. The nucleic acid in this digested sample was then acid phenol-chloroform (Ambion, Austin, TX) extracted, ethanol precipitated at -80°C overnight, and centrifuged at 16,000 x g to produce a DNA-free RNA pellet. RNA quality was assessed via agarose gel electrophoresis and RNA concentration determined by UV spectrophotometry using a Shimadzu UV-1650 (Kyoto, Japan).
Label
Cy3
Description
Sample compares mRNA expression in Caulobacter strain CB15N grown in M2-Glucose versus M2-Inositol.
Data processing
The mean background was subtracted from the mean signal for each channel. The background-subtracted Cy5 signal was divided by the background-subtracted Cy3 signal, and the log base 2 of this ratio was used as the value in the tables.
