Water was collected using Niskin bottles mounted on a CTD rosette. Biomass for metatranscriptomic analysis was collected by filtration (after a 200 micron pre-filtration) onto 47 mm, 1.2-micron pore size polycarbonate filters at <5 psi for no longer than 15 min to minimize degradation. Filters were flash frozen in liquid nitrogen and stored at -80 degrees C. Upon analysis, filters were thawed and RNA was extracted using TRIzol reagent according to the manufacturer’s protocol (Life Technologies). Metatranscriptome libraries were constructed using 500 ng of total RNA and a TruSeq RNA Sample Preparation Kit (Illumina; San Diego, CA) following the Low-Throughput protocol. The mean size of the final libraries was confirmed to be between 359-420 base pairs (bp) using an Agilent Bioanalyzer 2100 (Santa Clara, CA). Libraries were paired-end sequenced (2x150 bp) on the Illumina HiSeq platform. ORFs were annotated via BLASTP alignment (e-value > 10-3) to a comprehensive protein database, phyloDB, as well as screened for function de-novo by assigning Pfams, TIGRfams and transmembrane tmHMMs with hmmer 3.0 (http://hmmer.org/). PhyloDB 1.076 consists of 24,509,327 peptides from 19,962 viral, 230 archaeal, 4,910 bacterial, and 894 eukaryotic taxa. It includes peptides at KEGG, GenBank, JGI, ENSEMBL, CAMERA, and various other repositories, as well as from the 410 taxa of the Marine Microbial Eukaryotic Transcriptome Sequencing Project. Taxonomic annotation of ORFs was also conducted via BLASTP to phyloDB.

All cruise related data are available publicly at the Biological and Chemical Oceanography Data Management Office under project number 550825 (https://www.bco-dmo.org/project/550825). The metatranscriptomic data have been deposited in the NCBI sequence read archive (BioProject accession no. PRJNA528986: BioSample accession nos. SAMN11263616 - SAMN11263639 and SAMN11258802-SAMN11258825). Assembled contigs used in this study can also be found at https://scripps.ucsd.edu/labs/aallen/data/.

Unassembled reads and rRNA data from this study: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA528986
Assembled contigs: See Supplemental Files.