K2HPO4 was added to a final concentration of 1.0 mM for high phosphate medium (HPM) 0.2 mM for low phosphate medium (LPM)
Growth protocol
overnight cultures of Citrobacter rodentium were inoculated 1:50 into morpholinepropanesulfonic acid (MOPS) minimal medium containing 0.4% (w/v) 94 glucose, 0.01 mM Casamino acids, and 0.01 mM thiamine medium and 1.0 mM for high phosphate medium (HPM) or 0.2 mM for low phosphate medium (LPM) and grown until the OD600 was 0.7
Extracted molecule
total RNA
Extraction protocol
10mL of a culture was incubated with 20mL of RNAprotect solution (Qiagen) at room temperature for 15min. Cells were pelleted and RNA was purified using a FastRNA Pro Blue kit (Qbiogene). RNA samples were Dnase treated (Qiagen) and further purified using an RNeasy MiniElute kit (Qiagen)
Label
Cy3
Label protocol
5microg of total RNA was labeled with Cy3-ULS or Cy5-ULS (Kreatech) as per manufacturers instructions
K2HPO4 was added to a final concentration of 1.0 mM for high phosphate medium (HPM) 0.2 mM for low phosphate medium (LPM)
Growth protocol
overnight cultures of Citrobacter rodentium were inoculated 1:50 into morpholinepropanesulfonic acid (MOPS) minimal medium containing 0.4% (w/v) 94 glucose, 0.01 mM Casamino acids, and 0.01 mM thiamine medium and 1.0 mM for high phosphate medium (HPM) or 0.2 mM for low phosphate medium (LPM) and grown until the OD600 was 0.7
Extracted molecule
total RNA
Extraction protocol
10mL of a culture was incubated with 20mL of RNAprotect solution (Qiagen) at room temperature for 15min. Cells were pelleted and RNA was purified using a FastRNA Pro Blue kit (Qbiogene). RNA samples were Dnase treated (Qiagen) and further purified using an RNeasy MiniElute kit (Qiagen)
Label
Cy5
Label protocol
5microg of total RNA was labeled with Cy3-ULS or Cy5-ULS (Kreatech) as per manufacturers instructions
Hybridization protocol
Hybridization was conducted as per Agilent 2 color microarray-based gene expression analysis manual (version 5.7) with the modification 4 microg of labeled total RNA was used per hybridization.
Scan protocol
Images were quantified using Agilent Feature Extraction Software
