16S-rRNA-Seq of sea bulk water 6b (DNA)
DNA and RNA were co-extracted from the concentrated SBW, SML, and air samples as previously described by Lever et al. (2015) - https://doi.org/10.3389/fmicb.2015.00476. We amplified the hypervariable regions V3 and V4 of the 16S rRNA gene with the primers Bac341F (5-CCT ACG GGN GGC WGC AG-3) and Bac805R (5-GAC TAC HVG GGT ATC TAA TCC-3)from the DNA and cDNA samples.  The amplification of the 16S rRNA gene was performed according to the Illumina protocol, 16S Metagenomic Sequencing Library Preparation, Part # 15044223 Rev. B, with slight modifications. The modifications for the first PCR run were a thermal cycle of 20 for seawater samples and 30 for air samples. For the second PCR run, 10 l DNA template was used for the air samples, while 3 l template DNA was used for the seawater samples. The air samples were run for 15 cycles and the seawater samples for 10 cycles using the same conditions as for the first PCR. The third PCR mixture contained 2.5 l template DNA for both air and seawater samples, and the reaction was run for 8 cycles for both sample types using the same conditions as for the first two PCRs.  The final PCR products were quantified by using a Quant-iTTM dsDNA BR assay kit on a FLUOstar Omega fluorometric microplate reader (BMG LABTECH, Ortenberg, Germany). The PCR products were further diluted and pooled together to equimolar ratios, whereafter the pool was quantified using the Quant-iTTM dsDNA BR assay kit on a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, United States) and sequenced on the Illumina MiSeq platform (Illumina, San Diego, CA, United States).