organism: sludge microbes from anaerobic digesters digester id: co digester 2 organic loading rate: OLR=1.5 time points: Day 90 digester set: co digestion with dairy manure and poultry wast, OLR=1.5
Extracted molecule
genomic DNA
Extraction protocol
DNA was extracted from sludge samples using previously described protocols (Ma et al., 2015). Briefly, the sludge sample was resuspended in 630 µL DNA-extraction buffer, followed by treatment with 60 µL of a lysozyme mixture (37 °C, 60 min), 60 µL of a protease mixture (37 °C, 30 min), and 80 µL 20% sodium dodecyl sulfate (37 °C, 90 min). Treated sample suspension was subsequently extracted with the phenol–chloroform–isoamyl alcohol (25:24:1) at 65 °C for 20 min, and supernatant was extracted using the chloroform–isoamyl alcohol (24:1). DNA extract was then mixed with 0.6 volume of isopropanol and stored at 4 °C overnight. In addition, DNA was obtained by centrifuging the pellet, washing with 70% cold ethanol, drying and resuspension in nuclease-free water. DNA quality was evaluated with a NanoDrop spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA). Final DNA concentrations were quantified by PicoGreen, using a FLUO star Optima instrument (BMG Labtech, Jena, Germany). Purified DNA was stored at -80 °C.
Label
Cy3
Label protocol
As previously described (Yang et al 2013), DNA samples were labeled with the fluorescent dye Cy-3 using a random priming method and purified using the QIA quick purification kit (Qiagen, Valencia, CA, USA).
Hybridization protocol
The DNA was dried in a SpeedVac (ThermoSavant, Milford, MA, USA) at 45°C for 45 minutes. Then re-suspended in hybridization solution. GeoChip hybridization was carried out at 67 °C in an Agilent hybridization oven for 24 hrs.
Scan protocol
After slides washed, 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
co digestion with dairy manure and poultry wast, OLR=1.5
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 9 out of 21 samples.
