Wash buffer 1

The hybridization chamber was disassembled carefully and the microarray slide sandwich was completely submerged into wash buffer 1 (Agilent technologies, CA, USA) at room temperature. The slides were then gently pried open using a pair of forceps and the gasket was allowed to drop to the bottom of the jar. The microarray slide was quickly transferred to a slide rack submerged in wash buffer 1 and incubated for 5 minutes. Then the rack was transferred to the next dish containing wash buffer 2 (Agilent technologies, CA, USA) at 37°C for 1 minute. The slide rack was then transferred to a dish containing Acetonitrile for 1 minute followed by 30 seconds in Stabilization solution (Agilent technologies). The rack was removed carefully in order to minimize the number of droplets on the slide. The slides were scanned immediately on an Agilent scanner.

Genomic DNA was isolated using a method described by Pitcher et al. [42 (link)]. A total of 500 ng of genomic DNA from each Yersinia strain was fluorescently labeled with the BioPrime ArrayCGH labeling module (Invitrogen, Carlsbad, CA, USA) using either Cy3 or Cy5 (GE Healthcare, Buckinghamshire, UK). For each hybridization, one Cy3-labeled and one Cy5-labeled DNA sample were combined. The mixture was purified with a DNA purification kit (QIAquick PCR Purification Kit, Qiagen, Hilden, Germany) according to the manufacturer's instructions. The concentration of DNA and the incorporation of the dye were checked with the Nanodrop device (Nanodrop Technologies, Wilmington, MA, USA) before and after labeling. The differently labeled DNA sample pairs to be hybridized into one of the eight subarrays on each array slide were randomly selected.
A volume of 2.2 μL salmon sperm DNA (1 mg/mL) was added to 17.8 μL of labeled combined sample solution, and the mixture was heated at 95°C for 2 minutes for denaturation. A volume of 5 μL of 10x blocking agent (Agilent) and 25 μL of 2xGE (HI-RPI) hybridization buffer (Agilent) was added. A total of 45 μL of the solution was hybridized on each microarray at 65°C for 16 hours. The arrays were washed twice for 1 minute with Wash Buffer 1 (Agilent) and then for 1 minute with prewarmed Wash Buffer 2 (Agilent).

After the hybridization, microarrays and gaskets were disassembled in wash buffer 1 (Agilent Technologies). The slides were shifted to the wash buffer 2 after 05–30 min, (Agilent Technologies) and agitated at 37 °C for 01 min followed by the washing of slides with anhydrous acetonitrile. Scanning and image analysis were performed as per oligonucleotide array-CGH protocol (Agilent, version 4.0). Microarrays were scanned through Agilent Scanner (G2505C) and the data was extracted through Agilent’s Feature Extraction software (V.1.5.1.0).