Metagenome of Hatfield Marine Science Center ocean acidification mesocosm seawater
Seawater microbiome samples were collected from the intertidal pools that anemones resided in and from each of the lab treatment units on Days 0 and 20 of the experiment. To collect a microbiome sample from the seawater, a 50 mL syringe was flushed three times with seawater before being filled with 50 mL of seawater. Then a Sterivex GV 0.22 m filter unit (EMD Millipore Corporation, Billerica, MA, USA) was attached to the syringe and the seawater filtered through it. The ends of the filter unit were sealed with parafilm wax, the unit placed into a labeled reclosable poly bag, and then flash frozen in liquid nitrogen. All microbiome samples were subsequently stored at -80C until processed. Seawater microbiome samples were prepared for DNA extraction in a laminar flow hood following the protocol outlined in (Cruaud et al. 2017). Briefly, ethanol-sterilized pliers were used to aseptically remove the casing of the Sterivex filter cartridge. A sterile scalpel was used to cut away the filter panels which were placed into a sterile petri dish, cut into ~2 mm strips, and placed into a labeled ZymoBIOMICS ZR BashingBead Lysis Tube (Zymo Research, Irvine, CA, USA). These samples were immediately processed for DNA extraction. All microbiome genomic DNA samples were extracted using ZymoBIOMICS DNA Miniprep Kit (Zymo Research, Irvine, CA, USA) following the manufacturers instructions with the following modification: microbiome samples were vortexed on a Vortex Genie 2 Digital with TurboMix attachment (Scientific Instruments, West Palm Beach, FL, USA) for 20 minutes at max speed. DNA was extracted from sterile cotton swab-only and blank DNA extraction kit controls, and though no measurable DNA was detected, these samples were subsequently processed in the same way. Following DNA extraction, samples were run on a 1% agarose gel and through a Qubit dsDNA HS Assay Kit on a Qubit 3 (Invitrogen, Singapore) to check DNA quality and quantity respectively. DNA samples were diluted to the lowest sample concentration (14.5 ng  L-1) and then samples of the same treatment, symbiotic state, and same sampling day (e.g. Apo Field, high pCO2 Apo Day 0, low pCO2 Sym Day 20) were pooled together in equal proportions. Pooled samples were sent to the OSU Center for Genome Research and Biocomputing (CGRB) where they were prepared using the Nextera XT DNA library prep kit (Illumina, San Diego, CA, USA) for next generation sequencing. Following prep, libraries were analyzed using a High Sensitivity D5000 ScreenTape (Agilient Technologies Inc., Germany) and were then sequenced on two Illumina HiSeq 3000 lanes (Illumina, San Diego, CA, USA). The samples were randomly divided between the two lanes.