16S rRNA seq of IFC sub muros: oxic incubation
PCR amplification of the 16S rRNA V4 region using the primers 515F (5-GTG CCA GCM GCC GCG GTA A-3) and 806R (5-GGA CTA CHV GGG TWT CTA AT-3) (Caporaso et al., 2011) was performed using unique barcodes along with Illumina adapter sequences (Integrated DNA Technologies, Coralville, IA). Amplification was performed using a Mastercycler Nexus Gradient (Eppendorf, Enfield, CT) with a 3 min 94C hot start, followed by 30 cycles of: denaturing at 94C for 45 s, annealing at 50C for 60 s, and then a 72C extension for 90 s, followed by a final extension step at 72C for 10 min. The PCR products were gel purified and quantified using a Qubit dsDNA HS Assay Kit (Life Technologies, Waltham, MA). Samples were then sequenced on an Illumina MiSeq with paired end 250 bp reads.