README file for Dubinsky_2013_EST The MIAME information for this experiment is divided into the standard six sections: 1. The raw data for each hybridization: The raw data from each sample is in Affymetrix CEL file format. 2. The final processed data for the set of hybridizations in the study formatted as a data matrix used to draw the conclusions from the study. The values for each sample are trimmed mean (mean without the highest and lowest values) of the probe intensities for the perfectly-matched probes used to define each OTU. These values were scaled to an internal spike mix. Binary presence/absence scores were determined as described in Hazen et al. (Science, 2010, doi:10.1126/science.1195979). File name: MIAME_PhyloChip_data.xlsx 3. The essential sample annotation including experimental factors. File name: MIAME_metadata.xlsx 4. The experimental design including sample data relationships (e.g., which raw data file relates to which sample, which hybridizations are technical, which are biological replicates). Same file as above. 5. Reference commercial array catalog number. G3 Second Genome PhyloChip(TM) Assay: PN49-0002A 6. Essential laboratory and data processing protocols. The 16S rRNA gene was amplified using PCR with primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’) for bacteria and 4Fa (5’- TCCGGTTGATCCTGCCRG-3’) and 1492R for archaea. Each PCR reaction contained 1× Ex Taq buffer (Takara Bio Inc., Japan), 0.025 units/μl Ex Taq polymerase, 0.8 mM dNTP mixture, 1.0 μg/μl BSA, and 200 pM each primer and 0.15-0.5 ng genomic DNA as template. For the PhyloChip assay (PhyloTech Inc. San Francisco, CA) analysis each sample was amplified in 4 replicate 25 μl reactions spanning a range of annealing temperatures. PCR conditions were 95°C (3 min), followed by 30 cycles 95°C (30 s), 46-56°C (25 s), 72°C (2 min), followed by a final extension 72°C (10 min). Amplicons from each reaction were pooled for each sample, purified with the QIAquick PCR purification kit (Qiagen, Valencia, CA), and eluted in 20 μL elution buffer. PhyloChip microarrays have been described extensively (technical details, quality/processing controls, and reproducibility tests) by Hazen et al. (Science 2010). Oligonucleotide targets and probes on PhyloChip version G3 were synthesized by photolithography, representing publicly available bacteria and archaea 16S rRNA genes. Operational taxonomic units (OTUs) were defined as a group of highly similar 16S rRNA gene sequences (< 0.5% divergence). In total, there were 59,959 potential clusters, spanning 2 domains (archaea and bacteria), 147 phyla, 1,123 classes, and 1, 219 orders. The PhyloChip has been annotated according to the Greengenes taxonomy (Werner et al., ISME Journal, 2011, doi:10.1038/ismej.2011.82 and McDonald et al., ISME Journal, 2011, doi:10.1038/ismej.2011.139). For PhyloChip analysis, 500 ng of bacterial PCR product and 25 ng of archaeal PCR product were prepared for PhyloChip hybridization. PCR products were fragmented according to the previously described method (Brodie et al. AEM 2006) to a range of 50-200 bp as verified by agarose gels. Commercial kits were utilized for DNA labeling (Affymetrix WT Double Stranded DNA Terminal Labeling), and Affymetrix GeneChip Hybridization, Wash, and Stain kits were used for PhyloChip processing. Fragmented 16S amplicons and non-16S quantitative amplicon reference controls were labeled with biotin in 40 μL reactions containing: 8 μL of 5X TDF buffer, 40 units of TDF, and 3.32 nanomoles of GeneChip labeling reagent. After incubating at 37°C for 60 min, 2 μL of 0.5M EDTA was added to terminate the reaction. Labeled DNA was combined with 65 μL of 2X MES hybridization buffer, 20.4 μL of DMSO, 2 μL of Affymetrix control oligo B2, and 0.4 μL nuclease free water. Each reaction mixture was injected into the hybridization chamber of an array cartridge and incubated for 16 h in an Affymetrix hybridization oven at 48°C and 60 RPM. Hybridization solution was removed and the microarrays were stained according to the manufacturers instructions. Fluorescent images were captured with the GeneChip Scanner 3000 7G (Affymetrix, Santa Clara, CA). An individual array feature occupied approximately 8x8 pixels in the image file corresponding to a single probe 25mer on the surface. The central 9 pixels were ranked by intensity and the 75th percentile was used as the summary intensity for the feature. Probe intensities were background-subtracted and scaled to controlled Quantitative Standards (non-16S spike-ins) and outliers were identified as previously described (DeSantis et al., Microbial Ecology, 2007, doi:10.1007/s00248-006-9134-9). The hybridization score (HybScore) for an OTU was calculated as the mean intensity of the perfectly matching probes exclusive of the maximum and minimum.