Dataset: DNA and cDNA (RNA) 18S V4 tag sequences from 5m and DCM
Deployment: UpRISEE_SPOT_13-14

Methodology:

Seawater samples were collected from SPOT station (3333N, 11824W) from two depths. The SPOT station was sampled from 5m and DCM using 10 L niskin bottles mounted on a CTD rosette. The depth of the DCM was determined by in situ real-time fluorescence data from the CTD during each sample collection. Seawater samples were pre-screened through a 200 um nitex mesh and 80 um nitex using gravity filtration and and in-line filtration. Seawater collected at 5m (2L) and DCM (2L) were filtered onto a GF/F filter (nominal pore size 0.7 um; Whatman) and filters were stored in Caron Lab lysis buffer for DNA extraction and stored dry for RNA extraction. Filters were flash frozen in liquid nitrogen and stored in -80 C until further processing.
Total DNA extraction was accomplished by modifying the protocol described by DNeasy Plant Mini Kit (Qiagen, #69104). Filters were thawed for 5 min and 65C in a heat block and bead beaten by adding RNase-free silica beads and vortexed for 5 min with the addition of RNase A. Lysate was syringe-filtered and the appropriate proportions of neutralization buffer was added to the lysate. Total DNA was then extracted using DNeasy Plant Mini Kit, as per manufacturers instructions.
Total RNA was extracted using the RNeasy Mini Kit (Qiagen, #74104), as per manufacturers instructions. Filters were thawed on ice and 1.5 mls of RLT + (beta-Mercaptoethanol) was added to each filter and bead beaten by adding RNase-free silica beads and vortexed for 5 min. Genomic DNA was removed during RNA extraction (Qiagen, #79254) and checked for residual genomic DNA by performing PCR using (Stoeck et al., 2010) primers and observing an absence of amplification in an agarose gel. RNA was reverse transcribed into cDNA using iScript Select cDNA Synthesis Kit (BioRad, #1708896) with Random Primers. The resulting DNA and cDNA from each sample were PCR amplified using (Stoeck et al., 2010) primers with attached Illumina Adaptors (Forward- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-CASCYGCGGTAATTCC, Reverse- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-ACTTTCGTTCTTGATYRA). For each sample, we added 10 ng of genetic material to PCR tubes containing 0.5 um of each forward and reverse primer and 1X Q5 High Fidelity Master Mix for a final PCR volume of 25 ul and final concentration reaction mixture of .4 ng/ul. The PCR thermal profile had an initial activation step (Q5 specific) of 98C for 2 minutes, followed with 10 cycles of 98C for 10 seconds, 53C for 30 seconds, 72C for 30 seconds, and 15 cycles of 98C for 10 seconds, 48C for 30 seconds, and 72C for 30 seconds, and a final extension of 72C for 2 minutes. PCR products were purified using an AMPure bead clean up (Beckman Coulter #A63881). After purification, PCR products were normalized to one another and indexed using Illumina-specific P5 and P7 indices. Final indexes samples were pooled at equimolar concentration of 10 uM and sequenced using 250 bp PE sequences (Laragen).

The CTD was operated per Sea-Birds suggested methods, and was powered up and allowed to stabilize at approximately 0-5 meters prior to profiling. The instrument was lowered at 40 m per minute (maximum rate) for the entire depth of the sampling site (0-890 m).

Sampling ranges:
SPOT DNA samples were extracted from 2003-2018 (omitting 2017). Ten DNA samples were extracted for each month for two depths (5 m and DCM) from 2003-2018 (omitting 2017).
SPOT RNA samples were extracted from SPOT cruises from 2013 - 2016. Three years of RNA samples were collected for each month for two depths (5m and DCM) from 2013 - 2016. RNA samples were extracted for each month consecutively from 2013 - 2016; full dataset from 2013-2016 for RNA samples.

Location:
The SPOT station (33˚33 N, 118˚24 W) is located approximately 10 miles offshore from the Port of Los Angeles in the southern California Bight.