In August 2009, soil samples were collected from all of the plots. Five soil cores at a depth of 0-20 cm and diameter of 1.5 cm were randomly taken from each plot and mixed thoroughly to generate a soil sample representing the plot. Soil samples were kept on ice when transporting to laboratory, and sieved with 2 mm mesh to remove roots and stones. Soil samples were preserved at -80°C before DNA extraction.
Extracted molecule
genomic DNA
Extraction protocol
Soil genomic DNA was extracted using a FastDNA spin kit for soil (MP Biomedical, Carlsbad, CA, USA) following the manufacturer’s instructions. To purify it, DNA extract was mixed with 2.5 volume of 100% ice cold ethanol and 0.1 volume of 3 M NaOAc (pH 5.2) prior to overnight incubation at -20°C. DNA was precipitated by centrifugation for 30 minutes at 13,000 x g. Then supernatant was decanted and washed with 1 ml of 70% ethanol. DNA was air-dried and dissolved in 50 µl of nuclease-free water. DNA quality and quantity were measured using NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA) and with PicoGreen (Ahn et al 1996) using a FLUOstar Optima (BMG Labtech, Jena, Germany), respectively.
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
GeoChip data for soil sample collected at the 3400 m elevation, replicate 3 Soil microbes
Data processing
Signal intensities were quantified and processed using the data analysis pipeline as previously described (He et al 2010). Then processed GeoChip data were analyzed using the following steps: (i) remove the poor quality spots, which were flagged as 1 or 3 by ImaGene or with a signal to noise ratio (SNR) of less than 2.0; (ii) normalize the signal intensity of each spot by dividing the signal intensity by the total intensity of the microarray followed by multiplying by a constant; (iii) transform the data to the natural logarithmic form; and (iv) remove genes detected in only one out of three samples from the same elevation.
