Microarray-based Microbial Community Analysis

A phylogenetic microarray designed to target San Francisco Bay microbial communities (Figure S3, Table S1) included probes specific to ribosomal RNA operational taxonomic units (OTUs) as well as more general probes targeting the three domains of life (Bacteria, Archaea, Eukarya), two abundant marine bacterial orders (Alteromonadales and Rhodobacterales) and the genus Polaribacter[21] . Due to the variability in 16S diversity in different parts of the 16S phylogeny, there was no standard % similarity or taxonomic classification (genus, species, strain, etc.) that we could use to describe the lowest phylogenetic level targeted by the array. In general, taxa were targeted at the lowest possible phylogenetic level, subordinate to the genus level. To synthesize the microarrays, glass slides coated with indium-tin oxide (ITO; Sigma-Aldrich, St. Louis, MO, USA) were coated with silane Super Epoxy 2 (Arrayit Corporation, Sunnyvale, CA, USA) to provide a starting matrix for DNA synthesis. Custom-designed microarrays (spot size = 17 µm) were synthesized using a photolabile deprotection strategy [22] on the LLNL Maskless Array Synthesizer (Roche Nimblegen, Madison, WI, USA). Reagents for synthesis were delivered through an Expedite system (PerSeptive Biosystems, Framingham, MA, USA). For array hybridization, RNA samples (1 µg) in 1X Hybridization buffer (Roche Nimblegen, Madison, WI, USA) were placed in Nimblegen X4 mixer slides and incubated inside a Maui hybridization system (BioMicro Systems, Salt Lake City, UT, USA) for 18 hrs at 42°C and subsequently washed according to the manufacturer’s instructions (Roche Nimblegen, Madison, WI, USA). Arrays with fluorescently labeled RNA were imaged with a Genepix 4000B fluorescence scanner (Molecular Devices, Sunnyvale, CA, USA) at pmt = 650 units. Secondary ion mass spectrometry (SIMS) analysis of microarrays hybridized with ¹⁵N rRNA was performed at LLNL with a Cameca NanoSIMS 50 (Cameca, Gennevilliers, France). A Cs+ primary ion beam was used to enhance the generation of negative secondary ions. Nitrogen isotopic ratios were determined by electrostatic peak switching on electron multipliers in pulse counting mode, measuring ¹²C¹⁴N⁻ and ¹²C¹⁵N⁻ simultaneously. More details of the instrument parameters are provided elsewhere [21] . Ion images were stitched together and processed to generate isotopic ratios with custom software (LIMAGE, L. Nittler, Carnegie Institution of Washington). Isotopic ratios were converted to delta (permil) values using δ = [(R_meas/R_standard) –1]×1000, where R_meas is the measured ratio and R_standard is the ratio measured in unhybridized locations of the sample. All fluorescence and NanoSIMS data have been deposited to NCBI’s Gene Expression Omnibus archive under record number GSE56119.