Genomic DNA extraction as described in Berger et al. Journal of Bacteriology, February 2007, p. 1311-1321, Vol. 189, No. 4
Label
Cy5
Label protocol
Genomic DNA labelling as described in Berger et al. Journal of Bacteriology, February 2007, p. 1311-1321, Vol. 189, No. 4NA labelling and cDNA synthesis were carried out using the 3DNA Array 900 MPX Genisphere kit (Genisphere Inc., Hatfield, PA, USA) combined with the In situ Hybridization Kit Plus.
Genomic DNA extraction as described in Berger et al. Journal of Bacteriology, February 2007, p. 1311-1321, Vol. 189, No. 4
Label
Cy3
Label protocol
Genomic DNA labelling as described in Berger et al. Journal of Bacteriology, February 2007, p. 1311-1321, Vol. 189, No. 4NA labelling and cDNA synthesis were carried out using the 3DNA Array 900 MPX Genisphere kit (Genisphere Inc., Hatfield, PA, USA) combined with the In situ Hybridization Kit Plus.
Hybridization protocol
The hybridization conditions and the washing of the slide were performed as follows. To the 20 ul of DNA , 25 ul of control target Agilent, 60 ul of H2O and 105 ul of Agilent buffer were added. After 10 minutes of incubation at 80°C and 15 minutes at 65°C, the reaction was loaded on a pre-warmed slide in the hybridization chamber and incubated for 16 hours at 65°C, at four rpm in an Agilent oven. The cDNA hybridization was washed 10 min at 42°C in 6x SSC, 0.005% Triton x-100 and 10 min at RT in 0.2x SSC, 0.00016% Triton x-100. The 3DNA hybridization was carried out as described in the Genisphere protocol except for the hybridization volume which was increased to 204 l and the washing modified as follows : 10 min at 65°C in 2x SSC, 0.0016% Triton x-100, 5 min at RT in 2x SSC, 0.0016% Triton x-100 and 10 min at RT in 0.2x SSC, 0.00016% Triton x-100.
Scan protocol
The slides were scanned at 10 um with a Scanarray 4000 (Packard Biochip Technologies, Billerica, MA, USA)
Description
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Data processing
Data were extracted with Imagene 5.6 (Biodiscovery, El Segundo, CA, USA). Data were treated with homemade scripts in Python language (www.python.org) and a local installation of the ArrayPipe web server (Hokamp, K., et al. 2004. Nucleic Acids Res. 32:W457-W459). Probes showing a signal smaller than twice the standard deviation of the local background were considered without signal. Probes showing no signal or saturated signals in both channels were discarded from the analysis. The distribution of the log2-transformed signal ratios was analyzed for each hybridization reaction separately. The mean of a normal distribution fitting the main peak was calculated. The log2 signal ratio of each spot was then modified in order to shift the mean of the main peak of each hybridization reaction to 0.
