Genomic bacterial DNA was extracted following the protocol described in Maniatis Sambrook (Sambrook et al., 1989). DNA was sonicated in a Bioruptor (Diagenode, Liege, Belgium) to obtain fragments of approximately 500 bp.
Label
Cy3
Label protocol
Labelling was performed by overnight incubation at 37°C of 10 mg DNA with 50ml with 40 U of Klenow fragment (Fermentas), 5ml of 10 x buffer, 1 ml of Cyanine-3 labelled dUTP (Perkin Elmer, Schwerzenbach, Switzerland), 0.5 mM of dNTP (without dTTP) (Fermentas). Samples were purified using a PCR purification kit (Qiagen, Basel, Switzerland), and quality and concentration checked using a ND-1000 spectrophotometer (NanoDrop®, Witec AG, Littau, Switzerland).
Hybridization protocol
Prior to hybridisation, 3 mg of labelled DNA in 100 ml of 2 x Hybridisation buffer (NimbleGen® System, Inc., Madison, Wi, United States of America) and 40 ml Hybridisation Component A (NimbleGen® System, Inc.), 0.3 microl of 3’ labelled Cy3-CPK6 50 nM (Integrated DNA Technologies, Coralville, Ia, United States of America) and ddH2O up to 200 ml were denatured for 5 min at 95°C and cooled. Hybridisation was performed during 16 hrs at 4°C with a pump rate of 1ml/min in an aHybTM hybridisation station (Miltenyi Biotec, Bergisch-Gladbach, Germany). Washings were performed at 20°C in the station with NimbleGen® buffers I to III for 1 min each, respectively. The last washing buffer was incubated 1 min on the slide before blow-drying with high-pressure air.
Scan protocol
Slides were scanned at a gain of 500 to 600 at 532 nm wavelength and 5 microm resolution with a Genepix 4100A scanner (Axon Instrument, Sunnyvale, California, United States of America).
Description
Replicate 3 of 3. 227206-stew
Data processing
Signals of each slide were smoothed using the NMPP program (Wang et al., 2006). Normalisation per chip to 50th percentile and further analyses were performed in GeneSpring GX v7.3.1 (Agilent Technologies, Basel, Switzerland).
