Soil DNA was extracted using a PowerMax Soil DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA) according to the manufacturer’s protocol. DNA quality was assessed based on spectrometry absorbance at wavelengths of 230 nm, 260 nm and 280 nm detected by a NanoDrop ND1000 Spectrophotometer (NanoDrop Technologies Inc., now NanoDrop Products by Thermo Fisher Scientific). The 260/280 nm absorbance ratios were around 1.8, and 260/230 nm ratios were greater than 1.7. Finally, DNA was quantified with Pico Green using a FLUOstar OPTIMA fluorescence plate reader (BMG LabTech, Jena, Germany) and used for gene array labeling and sequencing library preparation.
Label
Cy3
Label protocol
For each sample, 1µg DNA was diluted to a volume of 25 μL and was mixed with 20 μL random primers (Invitrogen by Life Technologies, Grand Island, NY, USA), denatured at 99.9°C for 5 min and immediately chilled on ice. Then, 2.5 μL dNTP (5 mM dAGC-TP, 2.5 mM dTTP), 1 μL fluorescent dye Cy-3 dUTP (GE Healthcare UK Limited, Buckinghamshire, UK) and 80 U of the large Klenow fragment (Imer Inc., CA, USA) were added to the denatured DNA. The 50 μL mixture was incubated at 37°C for 6 hours, followed by heating at 95°C for 3 min. Labeled DNA was purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Cy-3 dye concentration was measured on a NanoDrop ND-1000 spectrophotometer (NanoDrop Products). The labeled DNA was then dried in a Savant SPD1010 SpeedVac concentrator system (Thermo Fisher Scientific) at 45°C for 45 min.
Channel 2
Source name
synthesized common oligonucleotide reference standard
sequence: 5′-CCGCACCTCGGACCGCACACAATCGTTTGAGGACGTGTAGCTGTGCTGGC-3′ molecule subtype: single stranded oligonucleotide
Extracted molecule
genomic DNA
Extraction protocol
Soil DNA was extracted using a PowerMax Soil DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA) according to the manufacturer’s protocol. DNA quality was assessed based on spectrometry absorbance at wavelengths of 230 nm, 260 nm and 280 nm detected by a NanoDrop ND1000 Spectrophotometer (NanoDrop Technologies Inc., now NanoDrop Products by Thermo Fisher Scientific). The 260/280 nm absorbance ratios were around 1.8, and 260/230 nm ratios were greater than 1.7. Finally, DNA was quantified with Pico Green using a FLUOstar OPTIMA fluorescence plate reader (BMG LabTech, Jena, Germany) and used for gene array labeling and sequencing library preparation.
Label
Cy5
Label protocol
For each sample, 1µg DNA was diluted to a volume of 25 μL and was mixed with 20 μL random primers (Invitrogen by Life Technologies, Grand Island, NY, USA), denatured at 99.9°C for 5 min and immediately chilled on ice. Then, 2.5 μL dNTP (5 mM dAGC-TP, 2.5 mM dTTP), 1 μL fluorescent dye Cy-3 dUTP (GE Healthcare UK Limited, Buckinghamshire, UK) and 80 U of the large Klenow fragment (Imer Inc., CA, USA) were added to the denatured DNA. The 50 μL mixture was incubated at 37°C for 6 hours, followed by heating at 95°C for 3 min. Labeled DNA was purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Cy-3 dye concentration was measured on a NanoDrop ND-1000 spectrophotometer (NanoDrop Products). The labeled DNA was then dried in a Savant SPD1010 SpeedVac concentrator system (Thermo Fisher Scientific) at 45°C for 45 min.
Hybridization protocol
The labeled DNA was resuspended in 10 µL hybridization solution containing 2.68 µL sample tracking control (Roche NimbleGen, Inc., Madison, WI, USA), 40% formamide, 25% SSC, 1% SDS, 1.6% Cy3 labeled alignment oligo, 1.6% Cy5-labeled alignment oligo and 2% Cy5-labeled common oligonucleotide reference standard target (NimbleGen). After denaturing at 95°C for 5 min, the mixtures were spun down and kept at 42°C to be deposited onto the glass microarray. GeoChip 4.2 slides were synthesized by NimbleGen in a 12-plex format (i.e., 12 arrays per glass slide). An HX12 mixer (NimbleGen) was affixed onto the array using a Precision Mixer Alignment Tool (NimbleGen). The array was preheated at 42°C in the 12-Bay MAUI Hybridization System (BioMicro Systems, Inc., Salt Lake City, UT, USA) for at least 5 min before the samples were loaded onto the array. Hybridizations were performed on the MAUI Hybridization System at 42°C in the presence of 40% formamide for about 16 hours with mixing. Then the mixers were removed from the slides while soaking in buffer I. The slides were manually washed in buffer I for 120 s, in buffer II for 60 s and in buffer III for 15 s and spun to dry in a NimbleGen slide spinner. All washing buffers were provided by NimbleGen.
Scan protocol
The dried microarray was scanned with a laser power of 100% and a photomultiplier (PMT) gain of 100% by a MS 200 Microarray Scanner (NimbleGen) at 532 nm and 635 nm. NimbleScan software version 2.5 (NimbleGen) was used to grid the images. After gridding, every spot (containing one unique probe) on the GeoChip array was fixed into a 7 × 7 pixel square and was adjacent to four blank equal-sized squares (void spaces). The probe signal was then calculated as the average intensity of the center 5 × 5 pixels for each spot, and the background noise signal was the average void intensity for each spot. The signal and background intensity report was generated also in NimbleScan software.
Description
Mo4D34
Data processing
Data normalization was completed on a web-based pipeline (http://ieg.ou.edu/microarray/) as follows. First, the raw probe signals, calculated based on the average pixel intensity of each probe, were adjusted based on control dye intensity and sample set total intensity. Second, signals were filtered by removing the spots with a signal to noise ratio less than 2, and the spots with signal intensity less than 2 times background. Third, probes that generated positive signals in less than 10 % of the samples from a given thaw site (all replicates from within each field site and along the depth profile) were considered low abundance genes and removed from that site for downstream analyses. We identified low abundance genes on a site basis instead of globally to retain functional gene probes unique to one site. Fourth, the probe signals were then normalized to represent relative abundance of genes in each sample. Unless otherwise specified, all gene abundance data reported in this manuscript represent relative abundance estimations based on the normalized signal intensities of probes targeting said gene.
