Genomic DNA was extracted from the about 5gm (wet weight) of sediment and 1.2L of water samples immediately after the samples were brought to the lab. Sediments were dewatered and DNA was extracted by a combination of mechanical, chemical and thermal lysis and chloroform-isoamyl alcohol purification using protocol from Zhou et al., 1996 (1). Water samples were filtered through sterile Millipore 0.22µm membrane filter (Thermo Fisher Scientific Inc.) to collect the biomass, followed by the DNA extraction protocol similar to the DNA extraction protocol for sediment samples. Humic substances were removed using OneStep™ PCR Inhibitor Removal Kit from Zymo Research Corporation (USA). The DNA quality and quantity was measured with PicoGreen using a FLUOstar Optima (BMG Labtech, Jena, Germany) before loading onto microarrays.
Label
Cyanine5
Label protocol
Please refer to He et al., 2010 (2), for detailed protocol. Briefly, 500 ng of genomic DNA from sediment and water samples, or about 3.0 µg of amplified DNA were labeled with the fluorescent dye Cy-5 using random priming method (Wu et al., 2006 (3)). Later, the whole amplified products were mixed with 20 μL random primers, denatured at 99.9 °C for 5 min, and then immediately chilled on ice. Following denaturation, the labeling master mix containing 2.5 μL dNTP (5 mM dAGC-TP, 2.5 mM dTTP), 1 μL Cy-5 dUTP (Amersham, Piscataway, NJ), 80 U of the large Klenow fragment (Invitrogen, Carlsbad, CA), and 2.5 μL water were added and then incubated at 37°C for 3 hours, followed by heating at 95 °C for 3 min. Labeled DNA was purified using the QIA quick purification kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions, measured on a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE), and then dried down in a SpeedVac (ThermoSavant, Milford, MA) at 45 °C for 45 min.
Hybridization protocol
Please refer to the He et al., 2010. The labeled target was re-suspended in 120 µl hybridization solution containing 50% formamide, 3 x SSC, 10 µg of unlabeled herring sperm DNA (Promega, Madison, WI), and 0.1% SDS, and the mix was denatured at 95°C for 5 min and kept at 50°C until it was deposited directly onto a microarray. Hybridizations were performed with a TECAN Hybridization Station HS4800 Pro (TECAN, US) according to the manufacturer’s recommended method. This equipment allows up to 48 hybridizations at one time, and well reproducible results were obtained.
Scan protocol
Please refer to the He et al., 2010. After washing and drying, the microarray was scanned by ScanArray Express Microarray Scanner (Perkin Elmer, Boston, MA) at 633 nm using a laser power of 90% and a photomultiplier tube (PMT) gain of 75%. The ImaGene version 6.0 (Biodiscovery, El Segundo, CA) was then used to determine the intensity of each spot, and identify poor-quality spots.
Data processing
Please refer to the He et al., 2010. Raw data from ImaGene were submitted to Microarray Data Manager at http://ieg.ou.edu/microarray/ and analyzed using the data analysis pipeline with the following major steps: (i) The spots flagged as 1 or 3 by Imagene and with a signal to noise ratio (SNR) less than 2.0 (He and Zhou, 2008 (4)) were removed as poor-quality spots; (ii) After removing the bad spots, normalized intensity of each spot was calculated by dividing the signal intensity of each spot by the mean intensity of the microarray; (iii) At least 0.34 time of the final positive spots (probes), or a minimum of two spots was required for each gene.
