Soil DNA was extracted by freeze-grinding mechanical lysis as previously described (Zhou et al 1996). Freshly extracted DNA was purified twice using 0.5% low melting point agarose gel followed by phenol-chloroform-butanol extraction. DNA quality and quantity were assessed by the ratios of 260 nm/280 nm and 260 nm/230 nm, and final DNA contents were quantified with a PicoGreen method using a FLUO star Optima .
Label
Cy3
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 mineral soil sample incubated at a near-freezing condition for 60 day, replicate 2
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.
