Dataset: Microbial eukaryotic diversity: Gorda Ridge SRA
Deployment: NA108

Sample collection:

The Gorda Ridge spreading center, ~200 km off the coast of southern Oregon was visited in May-June 2019 with the EV Nautilus (cruise NA108). Cruise operations were a collaborative effort with OET, NASA-SUBSEA, NOAA, URI, and WHOI. The ROV Hercules was positioned next to sites visually identified to have active hydrothermal flow. Temperatures at sites of active diffuse flow were obtained using a temperature probe controlled by the ROV Hercules arm. The SUPR sampler intake value then pumped this fluid into air-tight bags for grazing experiments and whole vent fluid water collection or pumped this fluid across 0.2 um PES Millipore filters (size, manufacturer). Fluid was also collected within the plume above each vent site by situating the ROV ~5 m above an active venting site and firing Niskin bottles. Background seawater from the water column at ~2,100 m was obtained by Niskin bottle. Samples from the plume and background collected by emptying Niskin bottles into acid-washed cubitainers and processed in the lab as described below. Upon recovery of ROV Hercules from each dive, bags filled with hydrothermal vent fluid were processed immediately. Filters were placed in RNAlater and kept frozen until return to the lab and fluid collected into bags was processed for grazing experiments (see 18S rRNA gene sequences) or filtered onto sterivex filters (both 18S and 16S sequences). 

16S sequences

DNA was extracted from 142 mm filters or Sterivex filters. The 142 mm filters were snap-frozen after folding and then rolling them into 50 mL tubes. To extract DNA, sterile forceps were used to pull the filter out of the tube, ethanol-flamed scissors were used to cut approximately 1/3 of the filter off and into a sterile 15 mL tube, then the rest of the filter was replaced at – 80oC in RNA Later. For the Sterivex filters, the filter housing was cracked open to expose the filter, the filter was cut off with sterile razor blade, and finally placed into a sterile tube with forceps. Approximately ½ of the filter was used, then the rest was saved at - 80oC covered in RNA Later.  

All filters were washed in 2 mL 1x sterile PBS by swishing them up and down in the PBS in the 15 mL tubes using sterile forceps. The PBS was transferred to 2 mL tubes and centrifuged at 14,000 rpm for 5 minutes to pellet cells. Filters were then placed in the pellet tubes for digestion. We added 900 uL DNA extraction buffer  (50 mL, 100mM Tris, 100mM EDTA, 100mM NaH2PO4, 1.5M NaCl, 1% CTAB), 20 uL proteinase K (10mg/mL), and 40 uL of lysozyme (50mg/mL) to each tube. We proceeded to do freeze-thaw 3 times at -80oC for 15 minutes and thaw at 37oC for 15 minutes. Tubes were then incubated in a 37oC heat block for 20 minutes. Filter-sterile SDS (50 mL of 20% solution in water) was added to teach tube and inverted several times to mix. Tubes were incubated for 2 hours in a 65oC water bath 

For the extraction steps, we worked in a fume hood and began by filling each tube to the top with phenol:chloroform:isoamyl alcohol (25:24:1, pH 8.0), and vortexing. Next, we centrifuged at low speed (3000 rpm) for 5 minutes and transferred the top layer to a new 2 mL tube, and repeated (total 2 washes). We added 0.6 volumes of room temperature 100% Isopropanol and inverted gently to mix. Tubes were incubated at room temperature overnight in a dark drawer and then centrifuged at top speed for 30 minutes at 4 οC. We then carefully pipetted off the buffer + isopropanol and saved this fraction in a new 2 mL tube. Ice cold 70% EtOH (1mL) was added to each 2 mL tube, inverted several times, and centrifuged at max speed for 10 min. EtOH was pipetted off and rinsing was repeated two times before allowing the tubes to air dry with a Kim wipe draped over the top. The dried DNA pellet was then dissolved into 50 uL nuclease-free sterile water and quantified using the PicoGreen Assay. 

DNA was PCR amplified using the 16S V4 primers 515F (Parada et al. 2016) and 806R (Apprill et al. 2015). Amplicons were sequenced with Illumina MiSeq sequencing. 

18S sequences

Samples collected for molecular analyses included in situ filters from the SUPR sampler, shipboard Sterivex filters, or time points from grazing experiments (all collected into 0.2-um pore size filters). Some of the 18S rRNA gene tag sequence samples are associated with grazing experiments, where vent fluid was incubated with analog prey (Fluorescently-labeled bacterial prey) to track grazing rates among single-celled microbial eukaryotes. These samples were taken at the start of the experiments (T0) or at various time points (T1, T2). 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.