Dataset: 18S rRNA gene tag sequences from DNA and RNA
Deployment: SPOT_Yellowfin_Cruises

Samples were collected seasonally at the San Pedro Ocean Time-series (SPOT) station at four depths (surface, subsurface chlorophyll maximum, 150 and 890 m). The SPOT station was sampled from 5 m, the subsurface chlorophyll maximum (SCM), 150 and 890 m using 10 L Niskin bottles mounted on a CTD rosette, during regularly scheduled cruises (https://dornsife.usc.edu/spot/).

Seawater from all samples was sequentially pre-filtered through 200 μm and 80 μm Nitex mesh to reduce abundances of multicellular eukaryotes (metazoa). Near-surface and SCM seawater (2 L) and 150 and 890 m seawater (4 L) was filtered onto GF/F filters (nominal pore size 0.7 μm; Whatman, International Ltd, Florham Park, NJ, USA) and immediately flash frozen in liquid nitrogen for later DNA and RNA extraction.

Total DNA and RNA were extracted simultaneously from each sample using the All Prep DNA/RNA Mini kit (Qiagen, Valencia, CA, USA, #80204). Genomic DNA was removed during the RNA extraction with RNase-Free DNase reagents (Qiagen, #79254). Total extracted RNA was checked for residual genomic DNA by performing a polymerase chain reaction (PCR) using DNA specific primers to ensure that no amplified products appeared when run on an agarose gel. RNA was reverse transcribed into cDNA using iScript Reverse Transcription Supermix with random hexamers (Bio-Rad Laboratories, Hercules, CA, USA, #170-8840).

The resulting cDNA and DNA from each sample were PCR amplified using V4 forward (5′ -CCAGCA[GC]C[CT]GCGGTA ATTCC-3′ ) and reverse (5′ -ACTTTCGTTCTTGAT[CT][AG]A-3′ ) primers (Stoeck et al. 2010). Duplicate PCR reactions were performed in 50 μL volumes of: 1X Phusion High-Fidelity DNA buffer, 1 unit of Phusion DNA polymerase (New England Biolabs, Ipswich, MA, USA, #M0530S), 200 μM of dNTPs, 0.5 μM of each V4 forward and reverse primer, 3% DMSO, 50 mM of MgCl and 5 ng of either DNA or cDNA template per reaction. The PCR thermal cycler program consisted of a 98◦C denaturation step for 30 s, followed by 10 cycles of 10 s at 98◦C, 30 s at 53◦C and 30 s at 72◦C, and then 15 cycles of 10 s at 98◦C, 30 s at 48◦C and 30 s at 72◦C, and a final elongation step at 72◦C for 10 min, as described in Rodrı ́guez-Martı ́nez et al. (2012). PCR products were purified (Qiagen, #28104) and duplicate samples were pooled. The ∼400 bp cDNA and DNA PCR products were quality checked on an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA).

Sampling Locations:
SPOT (33◦ 33′ N, 118◦ 24′ W) - surface, DCM, 150 m, and 890 m
Port of LA (33◦ 42.75′ N, 118◦ 15.55′ W) - surface
Catalina (33◦ 27.17′ N, 118◦ 28.51′ W)-surface

For protocols see:
https://www.protocols.io/view/sample-collection-from-the-field-for-downstream-mo-hisb4ee
https://www.protocols.io/view/rna-and-optional-dna-extraction-from-environmental-hk3b4yn
https://www.protocols.io/view/18s-v4-tag-sequencing-pcr-amplification-and-librar-hdmb246