sample type: soil mycrobial communities in metal polluted soils sample collection area: Miasteczko Śląskie transect
Extracted molecule
genomic DNA
Extraction protocol
DNA was extracted from 0.25 g soil using the PowerSoil DNA kit (MoBio Laboratories, Solana Beach, CA) following instructions of the manufacturer. DNA quality was evaluated by the absorbance ratios at A using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE). DNA quality was assessed by UV absorbance ratios of A260/A280 (> 1.8) and A260/A230 (> 1.7), values of 1.7–2.0 predict ‘clean DNA’, using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE), and DNA concentrations were measured with a PicoGreen method (Ahn et al., 1998).
Label
Cy5
Label protocol
As previously described (Yang et al 2013), DNA samples were labeled with the fluorescent dye Cy-5 using a random priming method and purified using the QIA quick purification kit (Qiagen, Valencia, CA, USA).
Hybridization protocol
Then DNA was dried in a SpeedVac (ThermoSavant, Milford, MA, USA) at 45°C for 45 minutes. The hybridization was carried out at 42°C for 16 hours on a MAUI hybridization station (BioMicro, Salt Lake City, UT, USA).
Scan protocol
After purification, GeoChip microarrays were scanned by a NimbleGen MS200 scanner (Roche, Madison, WI, USA) at 633 nm using a laser power and photomultiplier tube (PMT) gain of 100% and 75%, respectively.
Description
M2 GeoChip data for soil sample collected at twelve sites located along two distinct gradients of metal pollution in Southern Poland, replicate 1.
Data processing
Scanned images were processed using ImaGene version 6.1 (BioDiscovery, El Segundo, CA) by averaging the intensities of every pixel inside the target region (segmentation method). The mean signal intensity was determined for each spot, and the local background signals were subtracted automatically from the hybridization signal of each spot. The spot signals, spot quality, and background fluorescence intensities of scanned images were quantified with ImaGene. After this step all bad spots, which were flagged by the image processing software using predetermined criteria (defined as spots whose signals could not be accurately quantified due to their irregular shapes and/or contaminations) were removed and acceptable spots intensity data were extracted from ImaGene output files. Raw data obtained using ImaGene were uploaded to the laboratory’s microarray data manager and analyzed using a GeoChip 4.2 data analysis pipeline (http://ieg.ou.edu/microarray). Spots with a signal-to-noise ratio [SNR= (signal intensity-background intensity)/standard deviation of the background] greater than 2 were used for further analysis (including potential empty spots and good spots). Normalization was performed as described by He et al. (2007) and Liang et al. (2010). The hybridization signal was normalized by the mean signal intensity across all genes on the array. The across-array mean was calculated based on all intensities on the arrays after the removal of poor spots and outliers. Then, a ratio was calculated for each positive spot by dividing the signal intensity of the spot by the mean signal intensity to obtain the normalized ratio. The average signal intensities across all of the genes were expected to be approximately equal because the same amount of DNA was used for labeling and hybridization. A matrix was then generated from the normalized pixel intensities of all protein-encoding genes.
