Microbial DNA was extracted from the biofilm samples using the phenol chloroform method (Zhou, 1996) and purified using the QIAquick Gel Extraction Kit (QIAGEN Sciences, Germantown, MD, USA). The purified DNA was then quantified with a PicoGreen Kit (Eugene, OR, USA) and used for the GeoChip 4.0 hybridization
Label
Cy3
Label protocol
The DNA from each sample (500 ng) was labelled with the fluorescent dye Cy-3 (GE Healthcare, California, USA) by random priming (He et al. 2010)). The DNA was purified as explained above and dried in a SpeedVac (Thermo Savant, New York, USA)
Hybridization protocol
Each dried and labelled DNA sample was resuspended in 42 µL of hybridization solution. This solution consisted of 1 x HI-RPM hybridization buffer, 1 x comparative genome hybridization blocking agent, 0.05 µg・µL-1 Cot-1 DNA, 10 pM universal standard, and 10% formamide (final concentrations). The solution was later denatured by remaining at 95 ℃ for 3 min. To remove the bubbles created during the denaturation process, the conditions were maintained at 37 ℃ for 30 min. The hybridizations were carried out at 67 ℃ for 24 h
Scan protocol
The scanned images of the GeoChip hybridizations were obtained and converted by means of the Agilent Feature Extraction 11.5 software (Agilent Technologies, California, USA)
Data processing
The signal intensities, used as a proxy of abundances, were quantified and processed based on previous pipelines48 as follows: (i) removing probes with a signal-to-noise ratio of less than 2.0; (ii) normalizing the signal intensity of each probe by dividing the total signal intensity of a sample and multiplying by a constant; iii) removing singletons, i.e., genes detected only once on each mountain; and (iv) selecting those genes involved in the carbon (C), nitrogen (N), phosphorus (P), and sulphur (S) cycles and stress-related (St) processes
