[AT50-24 Bacterial 16S barcodes] - Bacterial 16S rRNA barcodes associated with methane seep invertebrates collected off of the Aleutian Islands, Gulf of Alaska on R/V Atlantis cruise AT50-24 from May to June 2024 (Collaborative Research: Redefining the footprint of deep ocean methane seepage for benthic ecosystems)

Specimens for molecular analysis were preserved within 2 h of collection in ~90% ethanol and stored at 4 °C. Total genomic DNA was extracted from whole specimens preserved in ethanol using the Qiagen DNeasy kit (Qiagen, Valencia, CA) according to the manufacturer's instructions.

Total DNA extracted was quantified via a Qubit 3.0 fluorometer using the dsDNA BR Assay Kit (Thermo Fisher Scientific). The V4-V5 hypervariable region of the 16S rRNA gene was PCR amplified using bacterial primers [515F: GTGYCAGCMGCCGCGGTAA and 806R: GGACTACHVGGGTWTCTAAT] with Illumina adapters on the 5′ end (San Diego, CA). Each PCR product was secondarily barcoded with Illumina NexteraXT index v2 Primers that included unique 8-bp barcodes, with NEB Q5 Hot Start High-Fidelity Mix at an annealing temperature of 66 °C for 11 cycles.