Samples were collected seasonally at the San Pedro Ocean Time-series (SPOT) station at four depths (surface, subsurface chlorophyll maximum, 150 and 890 m).\u00a0The 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/).
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Seawater from all samples was sequentially pre-filtered through 200 \u03bcm and 80 \u03bcm 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 \u03bcm; Whatman, International Ltd, Florham Park, NJ, USA) and immediately flash frozen in liquid nitrogen for later DNA and RNA extraction.
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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).
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The resulting cDNA and DNA from each sample were PCR amplified using V4 forward (5\u2032\u00a0-CCAGCA[GC]C[CT]GCGGTA ATTCC-3\u2032\u00a0) and reverse (5\u2032\u00a0-ACTTTCGTTCTTGAT[CT][AG]A-3\u2032\u00a0) primers (Stoeck et al. 2010). Duplicate PCR reactions were performed in 50 \u03bcL volumes of: 1X Phusion High-Fidelity DNA buffer, 1 unit of Phusion DNA polymerase (New England Biolabs, Ipswich, MA, USA, #M0530S), 200 \u03bcM of dNTPs, 0.5 \u03bcM 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\u25e6C denaturation step for 30 s, followed by 10 cycles of 10 s at 98\u25e6C, 30 s at 53\u25e6C and 30 s at 72\u25e6C, and then 15 cycles of 10 s at 98\u25e6C, 30 s at 48\u25e6C and 30 s at 72\u25e6C, and a final elongation step at 72\u25e6C for 10 min, as described in Rodr\u0131 \u0301guez-Mart\u0131 \u0301nez et al. (2012). PCR products were purified (Qiagen, #28104) and duplicate samples were pooled. The \u223c400 bp cDNA and DNA PCR products were quality checked on an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA).
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Sampling Locations:
\nSPOT (33\u25e6 33\u2032 N, 118\u25e6 24\u2032 W) - surface, DCM, 150 m, and 890 m
\nPort of LA (33\u25e6 42.75\u2032 N, 118\u25e6 15.55\u2032 W) - surface
\nCatalina (33\u25e6 27.17\u2032 N, 118\u25e6 28.51\u2032 W)-surface
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For protocols see:
\nhttps://www.protocols.io/view/sample-collection-from-the-field-for-downstream-mo-hisb4ee
\nhttps://www.protocols.io/view/rna-and-optional-dna-extraction-from-environmental-hk3b4yn
\nhttps://www.protocols.io/view/18s-v4-tag-sequencing-pcr-amplification-and-librar-hdmb246
Nucleotide bases with a Q score lower than 20 for the last 30 bp of each sequence were trimmed. Paired-end sequences were merged using FLASh (Magoc and Salzberg 2011) with a minimum of 10 bp and maximum of 150 bp overlap between each sequence pair. Sequences shorter than 350 bp, longer than 460 bp, or which had an average quality score lower than 25 were discarded using QIIME v1.8 (Caporaso et al. 2010). Chimeric sequences were identified and removed, by either de novo or reference-based chimera checking (identify chimeric seqs.py in QIIME, intersection method).\u00a0
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The code release v2 associated with this version of the dataset can be downloaded as a .zip file from the Supplemental\u00a0Documents section of this page. Future code updates will be accessible from the GitHub repository\u00a0https://github.com/shu251/V4_tagsequencing_18Sdiversity_q1.
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BCO-DMO Data Manager Processing Notes:
\n* File \"SRA_metadata_finalSHU.xlsx\" imported into the BCO-DMO data system.
\n* Added SRA_run_ids from file \"Listof_SRA_IDs.xlsx.\"
\n* added a conventional header with dataset name, PI name, version date
\n* modified parameter names to conform with BCO-DMO naming conventions
\n* blank values in this dataset are displayed as \"nd\" for \"no data.\"\u00a0 nd is the default missing data identifier in the BCO-DMO system.
\n* Split lat_lon column into \"lat\" and \"lon\" in decimal degrees.
\n* Added active html links to dataset for SRA Run IDs
\n*\u00a0 For curatorial purposes and to satisfy the OCE Sample and Data Policy requirements, the contributor's github was forked to BCO-DMO's github, a release was made, and the .zip file containing that release downloaded to BCO-DMO's data servers.\u00a0
\n\u00a0 \u00a0* BCODMO/V4_tagsequencing_18Sdiversity_q1 forked from shu251/V4_tagsequencing_18Sdiversity_q1
\n\u00a0 \u00a0* Release v2 created:\u00a0https://github.com/BCODMO/V4_tagsequencing_18Sdiversity_q1/tree/v2