Quantitative PCR of metazoan DNA from the ETNP St P2 in October 2019

Samples for eDNA were obtained from the Eastern Tropical North Pacific St P2 in October 2019 on the R/V Kilo Moana from 10 L Niskin bottles on a CTD rosette by vacuum filtering 4 L of water onto 47 mm diameter, 1 µm pore size cellulose nitrate membrane filters (Whatman). Water for biological replicates (n=2) was obtained from separate Niskin bottles on the same rosette. Samples were obtained at midday and near midnight on same day, on two separate occasions with an additional opportunistic midday cast included to fill in more depths in the vertical migration maximum region. Filters were immediately frozen at -80°C and were transported to HPL on dry ice. DNA was extracted using a CTAB Chloroform/Isoamyl extraction method (Renshaw et al., 2015). qPCR for the COI gene was performed using Leray primers (mlCOIintF: GGWACWGGWTGAACWGTWTAYCCYCC and jgHCO2198: TAIACYTCIGGRTGICCRAARAAYCA) (Leray et al., 2013) and SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) on a Bio-Rad C1000 Touch Thermal Cycler with a Bio-Rad CFX96 Touch Real-Time PCR Detection System for 45 cycles with an annealing temperature of 48°C. Each sample was run in triplicate. Additionally, in a separate well each sample was checked for PCR inhibitors by adding the sample to a known amount of zooplankton standard (1,050 pg/µL or 105 pg/µL; see below). A sample was determined to be inhibited if the Cq value of the standard was shifted later by 3 or more cycles. Inhibitors were only found in two samples (St P2: 32 and 52 m from the night). After dilution of the two samples by 1:10, PCR inhibition was ameliorated.These data support the paper "<b>Contributions of Vertically Migrating Metazoans to Sinking and Suspended Particulate Matter Fuel N</b>_{<strong>2</strong>}<b> Production in the Eastern Tropical North Pacific Oxygen Deficient Zone</b>."