Dataset: Microbial eukaryotic focused metatranscriptome data
Deployment: SPOT_Yellowfin_Cruises

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.

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’s Stranded mRNA library preparation kit using poly-A tail selection beads to select for eukaryotic mRNA (Kapa Biosystems, Inc., Wilmington, MA, #KK8420).

Also see:

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

The associated assembly files can be found at Zenodo (see Hu, S. K. (2017), DOI: 10.5281/zenodo.1202041).  The assembly files were also published in the journal publication Hu, et al. (2018).

Related code can be found in the github repository https://github.com/shu251/SPOT_metatranscriptome.  The version of the code used for these publications can be found in the Supplemental Files section of this page.