On the DeepCCZ cruise, benthic samples (sediments and nodules) were collected using the ROV Lu'ukai, using push corers and the ROV's manipulator arm. On the Abyssline cruises, benthic samples were collected with box corers and megacorers. On both cruises, water samples were collected with Niskin bottles mounted on a sampling rosette.

Sediment samples were sectioned into depth horizons and stored frozen at -80 degrees Celsius (C). Nodules were rinsed with 0.2-µm-filtered seawater and frozen whole at -80C. On DeepCCZ, seawater was sequentially collected on 3 µm and 0.2 µm pore-size filters; on the Abyssline cruises, seawater was collected on 0.2 micrometer (µm) pore-size filters. Filters were stored frozen at -80C.

Genomic DNA was extracted from seawater filters using a DNeasy Plant Mini Kit (Qiagen) with modifications as described in Shulse et al. (2017; doi: 10.1002/mbo3.428). Genomic DNA was extracted from subsamples of sediments and nodules using the FastDNA Spin Kit for Soil, modified as described in Shulse et al. (2017). gDNA was concentrated using the Zymo Clean & Concentrator-5 kit.

The V4-V5 region of the 16S rRNA gene was amplified using primers 515F-Y and 926R as recommended by Parada et al. (2016; doi: 10.1111/1462-2920.13023), with a multiplexing index on the forward primer following the design of the Earth Microbiome Project (https://earthmicrobiome.org/protocols-and-standards/16s/). Triplicate amplifications were combined and cleaned with an ENZA Cycle Pure Kit (Omega Bio-Tek) and then pooled at approximately equimolar proportions into two libraries. Libraries were sequenced at the University of Montana on an Illumina MiSeq using paired-end 250 v2 chemistry. Samples were demultiplexed by the sequencing facility.

Problem Report:
On DeepCCZ, sediments were not collected from the seamount in APEI 1 due to ROV constraints.