Seawater was collected from the San Pedro Ocean Time-series (SPOT) station off the coast of Southern California near the surface (5 m), 150 and 890 m, in late May 2015. Briefly, seawater was pre-filtered (80 mm) into 20 L carboys to minimize the presence of multicellular eukaryotes. Replicate samples (ranging in volume from 1.5-3.5 L) from each depth were filtered onto sterile GF/F filters (nominal pore size 0.7 mm, Whatman, International Ltd. Florham Park, NJ). While we cannot avoid some impact that sample handling (i.e., bringing samples to the surface) may have had on our results, filters were immediately placed in 1.5 mL of lysis buffer and flash frozen in liquid nitrogen in < 40 min and away from light to minimize RNA degradation.
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Total RNA was extracted from each filter using a DNA/RNA AllPrep kit (Qiagen, Valencia, CA, #80204) with an in-line genomic DNA removal step (RNase-free DNase reagents, Qiagen #79254) (dx.doi.org/10.17504/protocols.io.hk3b4yn). Extracted RNA was quality checked and low biomass samples were pooled. Six replicates were processed and sequenced from the surface, while pairs of filters were pooled for either 150 or 890 m, yielding 3 and 4 replicates respectively (Supporting Information Table S1). RNA concentrations were normalized before library preparation (Supporting Information). ERCC spike-in was added before sequence library preparation with Kapa\u2019s Stranded mRNA library preparation kit using poly-A tail selection beads to select for eukaryotic mRNA (Kapa Biosystems, Inc., Wilmington, MA, #KK8420).
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Also see:
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https://www.protocols.io/view/sample-collection-from-the-field-for-downstream-mo-hisb4eehttps://www.protocols.io/view/rna-and-optional-dna-extraction-from-environmental-hk3b4yn
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The associated assembly files can be found at Zenodo (see Hu, S. K. (2017), DOI:\u00a010.5281/zenodo.1202041).\u00a0 The assembly files were also published in the journal publication Hu, et al. (2018).
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Related code can be found in the github repository https://github.com/shu251/SPOT_metatranscriptome.\u00a0 The version of the code used for these publications can be found in the Supplemental Files section of this page.
Seawater was collected via Niskin bottles mounted with a CTD from the San Pedro Ocean Time-series (SPOT) station off the coast of Southern California near the surface (5 m), 150 and 890 m, in late May 2015. Raw sequence data was generated as part of a metatranscriptome study targeting the protistan community. Raw sequences are available at the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database (SRA Study ID: SRP110974, BioProject: PRJNA391503).
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These data were published in Hu et al. (2018).
Sequence adapters, low quality (phred score < 10, from 5\u2019 and 3\u2019 ends, and within a 25 bp sliding window) or short sequences (< 50 bps), and sequences containing more than 50 consecutive As or Ts were removed using Trimmomatic v. 0.32 (Bolger et al., 2014). All quality trimmed sequences were aligned to ERCC sequences using \u2018align_and_estimate_abun- dance.pl\u2019 in the Trinity v. 2.1.1 (Grabherr et al., 2011) package. Reads that were aligned to ERCC sequences were removed using a custom PERL script (available: https://github.com/shu251/SPOT_metatranscriptome).