Sludge samples were collected on the monthly basis from during October, 2012 to September, 2013. Briefly, 2 liters of each sludge sample were obtained near the outlets of anaerobic tanks, stored in a portable dry ice container and immediately transported to laboratory. A small part of each sample was kept at 4°C for physicochemical properties analysis, while others were precipitated by centrifugation at 14,000 g for 10 minutes. After centrifugation, the pellet was air dried for 30 min and stored at -80°C for DNA extraction, while the supernatant was decanted. It was noted that the solid content of all 48 samples was less than 10%. DNA was extracted by MoBio PowerSoil DNA isolation kit (MoBio Laboratories, Carlsbad, CA, USA) and then purified by agarose gel electrophoresis followed by extractions with phenol and chloroform, and precipitation with butanol. After purifying, NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) were applied to access DNA quality, confirming that the A260/A280 ratio was between 1.8 to 2.0 and A260/A230 over 1.5.
Label
Cy5
Label protocol
Purified DNA was first labeled with the fluorescent dye Cy-3 (GE Healthcare, CA, USA) using random priming as described previously (Cong et al., 2015), then purified by a QIAquick Purification kit (Qiagen, Valencia, CA, USA), and dried in a SpeedVac (Thermo Savant, NY, USA) at 45 °C for 45 min. The dried DNA was then resuspended into ??? μl of DNase/RNase-free distilled water and evenly mixed with ??? μl of hybridization solution, which contains 1 × Acgh blocking, 1 × HI-RPM hybridization buffer, 10 pM universal standard DNA, 0.05 μg/μl Cot-1 DNA, and 10% formamide (final concentrations).
Hybridization protocol
Subsequently, the mixed solution was kept at 90 °C for 3 min for denaturation, and incubated at 37°C for 30 min before GeoChip hybridization was carried out at 67 °C for 24 hrs with a rotation at 20rpm in Agilent hybridization oven.
Scan protocol
After hybridization, Agilent Wash Buffers were applied to wash away unbounded DNA at ordinary temperature, and the arrays were scanned with a NimbleGen MS200 Microarray Scanner (Roche NimbleGen, Madison, WI, USA) at 633 nm. Eventually the images data were extracted by Agilent Feature Extraction program.
Description
GeoChip data for sludge sample collected in Ninghai, thermophilic tank, February
Data processing
The raw microarray data was analyzed by GeoChip Microarray Data Manager pipeline (http://ieg.ou.edu/microarray/) as previously described (Xu et al., 2010). We processed raw GeoChip data using the following steps: (i) remove the spots with a signal-to-noise ratio (SNR) less than 2.0; (ii) log-transformed the data and then, on each microarray, divide them by the mean intensity of all the genes; and (iii) remove genes detected only once in each digester. Finally we acquired GeoChip data for further analysis.
