Sample collection from Axial Seamount:

From Fortunato et al (2018)

Sample collection from Axial Seamount took place in September 2013, August 2014, and August 2015. ROVs ROPOS and JASON were used to collect diffuse hydrothermal venting fluid. At each site, 3-L of diffuse venting fluid was pumped (100-150-ml per minute) onto a 0.22-um 47mm GWSP filter (Millipore). This fluid was collected using the Hydrothermal Fluid and Particle Sampler (HFPS; Butterfield et al. 2004), which was mounted on an ROV. The fluid intake for the HFPS has a temperature sensor to ensure a constant temperature during fluid collection. Filters were preserved in situ with RNALater. Background seawater was collected from a depth of 1500-m using a CTD mounted with 10-L Niskin bottles.

Extraction and sequencing for 18S tag-sequencing:

For all samples, RNA was extracted and amplified similarly to the protocol described in Hu et al. 2018 (https://dx.doi.org/10.17504/protocols.io.hk3b4yn). Frozen filters were thawed and placed into sterile 15-ml falcon tubes with sterile forceps, 1-2 mL of RLT+ buffer (with β-Mercaptoethanol, Qiagen, Valencia, CA, USA) and RNase-free silica beads was added to each tube. Falcon tubes were bead-beaten by vortexing vigorously for 5 minutes. The original sample collection tubes with RNAlater were centrifuged to pellet any cellular material left in the RNAlater; the RNAlater was removed and replaced with 500-ul of RLT+ buffer (with β-Mercaptoethanol). This was vortexed and added to the 15-ml falcon tube. RNA was extracted with the RNAeasy kit (Qiagen #74104) with the in-line genomic DNA removal step (RNase-free DNase reagents, Qiagen #79254). RNA concentrations were determined using the Ribogreen protocol. Extracted RNA was reverse transcribed into cDNA using a cDNA synthesis kit (iScript Select cDNA Synthesis, BioRad, #1708896, Hercules, CA); the concentration of RNA was normalized for the cDNA synthesis reaction (input –ng of RNA). Primers targeting the V4 hypervariable region of the 18S rRNA gene (Stoeck et al. 2010; Hu et al. 2015) were used in PCR reactions, which consisted of a final concentration of 1X Q5 High Fidelity Master Mix (NEB #M0492S, Ipswich, MA), 0.5 μM each of forward and reverse primers, and 1 ng of genetic material. The PCR thermal protocol started with an initial activation step (Q5 specific) of 98°C for 2 min, followed with 10 cycles of 98°C for 10 s, 53°C for 30 s, 72°C for 30 s, and 15 cycles of 98°C for 10 s, 48°C for 30 s, and 72°C for 30 s, and a final extension of 72°C for 2 min (modified from Rodriquez Martinez et al. 2012). The original extract total RNA was also PCR amplified to ensure no genomic DNA was present in the sample. PCR products were checked by confirming the presence of an ~400 bp product on an agarose gel. In cases with no amplification, the PCR reaction was repeated with a higher concentration of cDNA (1.5-2 ng). If this did not yield the expected PCR product, the reaction was repeated with an additional 5 cycles. Three shipboard blanks (MilliQ water) and one extraction blank were also extracted and PCR amplified; while no PCR product was observed in these control samples they were processed similarly to all true samples and sequenced. All PCR products were cleaned using the AMPure bead clean up (Beckman Coulter #A63881, Brea, CA). Samples were multiplexed, pooled at equimolar concentrations and sequenced using the MiSeq 300 x 300 bp PE sequencing at Marine Biological Laboratory Bay Paul Center sequencing facility.