title: Metabarcoding of abyssal zooplankton
design: DNA metabarcoding on the recombined bulk community sample was performed using a modified protocol for the E.Z.N.A Blood Maxi Kit (OMEGA Bio-Tek). All sorted specimens were reconstituted into a total community sample on a clean 63-µm Nitex filter via a polycarbonate filter holder and gentle hand-operated vacuum pump. Plankton was flushed with dH2O to reduce the transfer of EtOH. All animals were transferred to a clean 50mL tube by backflushing with 0.5 mL of CSPL buffer (Omega Bio-Tek, Georgia, USA). Zooplankton were finely ground by pestle and 4.5 mL of additional CSPL buffer was added with 250µL of Proteinase K. Samples were vortexed and incubated overnight in a 55°C water bath. Subsequent steps followed the manufacturer's protocol. Additional modifications included warming the elution buffer to 70°C to enhance DNA recovery, and minimizing the elution volume to 1.1 mL for the first elution. The first elution was concentrated using the DNA Clean & Concentrator kit (Zymo Research). The V1-V2 region of the nuclear 18S rRNA gene (18S V1-V2) using primers F04: 5' GCTTGTCTCAAAGATTAAGCC 3' and R22mod: 5' CCTGCTGCCTTCCTTRGA 3' (∼365 bp; (Fonseca et al. 2010; Sinniger et al. 2016) was amplified using polymerase chain reaction (PCR). Each reaction contained 3 µL of template DNA, 12.5 µL of MyFi™ Mix (Meridian Bioscience), 1 ng/µL Bovine Serum Albumin (0.5 µL), and 10 µM forward and reverse primers (0.5 µL each), with molecular grade H20 added for a total reaction volume of 25 µL. PCR conditions were initial denaturation at 95 °C (2 min), followed by 40 cycles of denaturation at 95 °C (45 sec), annealing (55 °C for 45 sec), and extension at 72 °C (1 min), followed by final extension at 72 °C (10 min). PCR amplification was conducted in duplicate and products were pooled for library preparation. Pooled amplicons were purified using AMPure® XP beads (Beckman Coulter®), quantified using a Qubit® 1X dsDNA High Sensitivity Fluorescence kit (Thermofisher Scientific), and normalized to a concentration of 4ng/µL with molecular grade H20. Aerosol barrier pipette tips were used throughout DNA extraction, PCR, and library, additionally filters, filter holders and tissue grinders were treated with 10% bleach overnight, 3x dH2O rinsed, and dried prior to use to minimize contamination."