Bicarbonate was added to a final concentration of 45mM
Growth protocol
overnight cultures of E.Coli EHEC EDL933 were inoculated into LB medium 1:100 and grown until the OD600 was 0.85-0.95
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
Channel 2
Source name
liquid culture of Escherichia coli EHEC EDL933 Δstx1-2
Bicarbonate was added to a final concentration of 45mM
Growth protocol
overnight cultures of E.Coli EHEC EDL933 were inoculated into LB medium 1:100 and grown until the OD600 was 0.85-0.95
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
