Dataset: Lost City hydrothermal fluids sequence data
Deployment: AT42-01

Methods and Sampling 

On the seafloor, venting fluids were slowly pumped through 0.2 μm Millipore Sterivex cartridge filters or into acid-washed Kynar bags with the HOG sampler. Samples intended for RNA extractions were collected into 2 L Kynar bags containing 67 mL of a stop solution (97.5% 200 proof ethanol, 2.5% Trizol LS). Fluid temperatures were monitored in real-time during sampling with a probe embedded into the sampler intake. Immediately upon shipboard recovery of ROV Jason, all Sterivex filters were stored at -80 ºC.

Extraction of DNA from all Sterivex filters (including in situ-filtered fluids and fluids collected in Kynar bags and filtered shipboard) was conducted as described in the full protocol available via the Zenodo-archived GitHub repository (DOI: 10.5281/zenodo.5798015) and on protocols.io (DOI: dx.doi.org/10.17504/protocols.io.bykqpuvw). Total RNA was extracted from the Sterivex filters with a modification of the DNA extraction protocol optimized for RNA. The full protocol is available in the Zenodo-archived GitHub repository (DOI: 10.5281/zenodo.5798015) and on protocols.io (DOI: dx.doi.org/10.17504/protocols.io.bykspuwe). First-strand synthesis of cDNA was performed with SuperScript IV Reverse Transcriptase and random hexamers (Thermo Fisher).

Sequencing of amplicons generated from 16S rRNA genes and cDNA was performed at the Genomics Core Facility at Michigan State University on an Illumina MiSeq instrument using dual-indexed Illumina fusion primers targeting the V4 region of the 16S rRNA gene. Metagenome libraries were constructed with size-selected, sonicated DNA fragments of 500-700 bp with the NEBnext Ultra DNA II library kit for Illumina (E7645S). Paired-end sequencing (2 x 125 bp) of metagenomic libraries was conducted at the University of Utah High-Throughput Genomics Core Facility at the Huntsman Cancer Institute with an Illumina HiSeq2500 platform. The two metatranscriptome libraries were constructed and sequenced by the University of Utah High-Throughput Genomics Core Facility at the Huntsman Cancer Institute. Total RNA was hybridized with NEBNext rRNA Depletion Solution Bacteria (E7850L) to substantially diminish rRNA from the samples. Stranded RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina (E7770L). Following the transfer of the flowcell to an Illumina NovaSeq 6000 instrument, a 150 cycle paired-end sequence run was performed using a NovaSeq 6000 S4 reagent Kit v1.5 (20028312).