Dataset: Lau Basin and Loihi Seamount microbial mats
Deployment: TN225

Samples were collected with a closable PVC scoop sampler that was designed to sample mat material along with bottom waters while minimizing cross-contamination from other samples or with ambient seawater. The sampler was constructed with 3" PVC pipe and was washed with 70% ethanol before being rinsed and filled with filter-sterilized deionized water and sealed with a ball valve before deployment. The valve was opened immediately prior to sampling and closed directly after the sample collected and remained sealed until the sample was recovered on the ship. All samples were allowed to settle at 4 degrees C for approximately 2 hours before the overlying seawater was decanted and the mat material frozen at -80 degrees C until DNA was extracted in the lab.

Note: The J2-373 dive video can be found on the virtual van (http://4dgeo.whoi.edu/jason/).

DNA extraction: Total genomic DNA (gDNA) was extracted from each sample in duplicate using the FastDNA Spin Kit for Soil following the manufacturer's protocol (Qbiogene, Irvine, CA). Extracted gDNA from each sample was pooled, cleaned, and concentrated using Montáge PCR centrifugal filter devices (Millipore, Bedford, MA). The gDNAs were then quantified using a Nanodrop ND-1000 spectrophotometer and were diluted to 10 ng DNA/ml using filter sterilized 10 mM Tris 0.1 mM EDTA (pH 8.0).

Metagenome sequencing: Two micrograms of gDNA was sheared using a Covaris S2 sonicator and 400bp fragments were size selected for library construction. The library was constructed using the TruSeq DNA sample prep kit (Illumina) following the manufactures protocol. Sequencing was performed using an Illumina HiSeq 2000 sequencer.