7 protocols
AccessionType
normalization data transformation protocol
Data from the five slides were normalized by a Lowess fit using GProcessor. Using this program we calculated the mean of log2 ratios and median of log2 ratios across all five slides; and used the median of ratios for further analysis. Data was merged using GEPAS and a t-test was performed by GProcessor to select genes that were differentially expressed based on all good replicate spots. across five slides; only genes with p-values <0.01 were analyzed further. ID_REF = VALUE = Normalized log2 ratio representing reference/test (DW2/pFLP-Ec over DW2/pFLP-Bs)
array scanning protocol
GenePix Pro v.4.1 software (Axon Instruments, Inc.). Photomultiplier tube voltage was altered for the initial scanning to ensure proper channel balance and to decrease background.
normalization data transformation protocol
Data from the five slides were normalized by a Lowess fit using GProcessor. Using this program we calculated the mean of log2 ratios and median of log2 ratios across all five slides; and used the median of ratios for further analysis. Data was merged using GEPAS and a t-test was performed by GProcessor to select genes that were differentially expressed based on all good replicate spots. across five slides; only genes with p-values <0.01 were analyzed further. Raw data is not available for this sample. ID_REF = SL44-Log2-Channel1(635) = SL44-Log2-Channel2(532) = VALUE = Normalized log2 ratio representing reference/test (DW2/pFLP-Ec over DW2/pFLP-Bs)
hybridization protocol
cDNA from DW2/pFLP-Bs and DW2/pFLP-Ec were cohybridized to slides containing 70-mer oligos representing 4,290 annotated ORFs in E. coli MG1655. The hybridization solution (20 μl of cDNA mixture, 30 μl of 2X SDS based hybridization buffer, 3 μl of LNA dT Blocker, 8 μl of nuclease-free water) was placed over a pre-warmed microarray slide and covered with a Corning 22 x 60 mm coverslip. The slide was then sealed in a hybridization chamber (Genehyb) and submerged for 2-3 days in a 58°C water bath. Microarray slides were washed for 10 min at room temperature starting with pre-warmed (42°C) 2X SSC (Ambion) followed by 0.2% SDS, 10 min in 2X SSC at room temperature, and lastly 10 min in 0.2X SSC at room temperature. Slides were dried by centrifugation at 1000 rpm, 2 min. For the second hybridization to allow the 3DNA capture reagent to bind to the complementary Cy3- or Cy5-specific 3DNA capture sequences ligated to the cDNAs, each slide received 60 μl of hybridization solution (2.5 μl of Cy3 capture reagent, 2.5 μl of Cy5 capture reagent, 30 μl of 2X SDS-based hybridization buffer, 25 μl of nuclease-free water) and was hybridized as before by submersion for 3 to 4 hrs in a 65°C water.
labelling protocol
The synthesis, labeling of cDNA with Cy3 or Cy5, and hybridization to microarray slides were performed using the 3DNA Array 900MPX Expression Array Detection Kit (Genisphere) with 2 μg of total RNA for each strain.
nucleic acid extraction protocol
Total RNA extracted using phenol:chloroform method followed by two DNase I treatments (Qiagen DNase I and Ambion Dnase Turbo).
growth protocol
From a glycerol stock, cells were streaked onto LB-agar plates supplemented with kanamycin. Using a sterile toothpick a single colony was picked and placed into 100 ml of LB broth with kanamycin. Optical readings were taken and cells were pelleted for RNA harvest when the liquid culture reached an OD of 0.5