All samples were collected via ROVs, where an intake valve was positioned at the location of active diffuse flow (<100C) and collected into bags, bottles, or a filter directly. If collected into a bag or bottle, fluid was filtered immediately upon recovery. All filters had a pore size of 0.2µm and were composed of PES material.

Samples collected at the Mid-Cayman Rise and the Axial Seamount locations utilized ROV Jason; at the Mid-Cayman Rise fluid was collected with the HOG sampler, the SUPR sampler was used to collected fluids at Axial Seamount. Samples collected at the Gorda Ridge used ROV Hercules with the SUPR sampler.

Metatranscriptomics

Messenger RNA (mRNA) was subset from previously extracted RNA from all sites and grazing experiments (Qiagen RNA mini kit). Then metatranscriptome libraries were prepared from the mRNA. Library was prepped with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina #E7760S; with a Poly(A) mRNA magnetic isolation module first. For fragmentation, samples were incubated at 94C for 10 minutes. PCR cycles done until enough was isolated, range of 16-28. Mainly 20 or 22 cycles total. Input total RNA for metatranscriptome libraries was either 100 ng or 10 ng. However, with mRNA content ranging from 1-5% of the total RNA, the PCR amplification steps needed to be modified for the estimated input mRNA.

Libraries were sequenced with NovaSeq at the Northwest Genomics Sequence Center (Seattle, WA). Sequenced with NovaSeq S2 300 cycles. An average of 100 million sequences per sample were recovered from the metatranscriptome sequencing.

Metagenomics

Duplicate samples (other half of filter) from in situ sites at Mid-Cayman Rise were extracted for DNA (n = 12), then prepared as metagenome libraries, and sent for NovaSeq sequencing (data received January 2023). DNA extracted with MasterPure Complete DNA and RNA Purification Kit (Lucigen MC85200). Input DNA diluted to 50 ng total, or all DNA input (For low concentration samples) and sheared to target 400 bps with Covaris M220 focused-ultrasonicator, utilizing SonoLab 7.2. For 70 sections, 200 cycles (bursts) at 50 watts (Peak incident power), duty factor = 10%, and average incident power was 5 watts. Min temp: 18C, setpoint: 20C, and max: 22C.

Libraries were prepped with Ovation Ultralow System V2 (from Nugen). Library amplification was done at 15 cycles, and pooled. Sequencing was done with a NovaSeq S2 300 Cycle (UW Genomics NWGC). Over 80 million sequences per sample were recovered, to ensure sufficient sequencing depth.