16S DNA of seawater samples from hydrothermal fluid addition experiments
DNA was extracted following the phenol-chloroform method (Sambrook and Russell, 2001). Primers 515F (GTGYCAGCMGCCGCGGTAA) (Parada et al., 2016) and 806R (GGACTACNVGGGTWTCTAAT) (Apprill et al., 2015) were used to target the V4 region of the bacterial 16S rRNA (~390 bp). Each PCR reaction (20 l) contained 10 l of AccuStart II PCR SuperMix (2x), 20 M of each primer, 3.8 l of water, and 5 l of DNA template. The PCR program comprised an initial denaturation step at 94C during 3 min, 35 amplification cycles, each consisting of 94C for 45 s, 50C for 60 s and 72C for 90 s, a final elongation step at 72C for 10 min, and cooling to 4C. On the other hand, the V4 region of the eukaryotic 18S rRNA (~26050 bp) was targeted using TAReuk454FWD1 (CCAGCASCYGCGGTAATTC) and TAReukREV3 (ACTTTCGTTCTTGATYRA) primer set (Stoeck et al., 2010). PCR reactions were set up as previously described for the 16S rRNA gene. On this occasion, the PCR program consisted of an initial denaturation step at 94C during 3 min, 15 amplification cycles of 94C for 45 s, 53C for 30 s and 72C for 30 s, followed by 20 additional amplification cycles, each consisting of 94C for 45 s, 48C for 30 s and 72C for 30 s, a final elongation step at 72C for 10 min, and cooling to 4C. For each library, amplified PCR products were cleaned, indexed, and quantified following the Illuminas 16S metagenomic sequencing library preparation protocol. Equimolar amounts of cleaned-up indexed samples were pooled and paired-end sequenced (2x300) on an Illumina MiSeq platform (Macrogen, Korea).