Bulk community metabarcoding sample (210-560 micron size fraction) on January 5th, 2023 from RES2.5 100-200 m
Total DNA was extracted from mixed zooplankton community samples using QIAGEN DNeasy Blood & Tissue Mini Kit (catalog # 69504) following manufactures protocols with an extended 24-hour proteinase K incubation. A 313-bp region of the mitochondrial cytochrome c oxidase subunit I (mCOI) gene was amplified using the Leray mlCOIintF and jgHCO2198 primers (mlCOIintF 5-GGWACWGGWTGAACWGTWTAYCCYCC-3; JGHCO2198 5-TAIACYTCIGGRTGICCRAARAAYCA-3) (Leray et al. 2013). Amplification of the targeted gene was done using PCR in 20-L volumes using the ultrahigh-fidelity ThermoFisher Invitrogen Platinum II Taq Polymerase and Hot-Start PCR Master Mix (catalog # 14000013) with 5 L of sample DNA normalized to 5 ng L-1. The PCR was run with an initial denaturation step at 94C for 3 min followed by 28 cycles of 94C for 15 s, 48C for 32 s, 68C for 15 s, and a final extension for 5 min at 68 C. Amplifications were quality-checked for amplicon size using gel electrophoresis. Samples were then purified, indexed, and pooled using Illumina Nextera Library Prep kit and sequenced using Illumina MiSeq (V3 300-bp paired end) at the University of Hawaii ASGPB facility (https://www.hawaii.edu/microbiology/asgpb/).