Pyramimonas tychotreta and Micromonas polaris (CCMP 2099) mixotrophy RNA-Seq

Replicate cultures (n=4 for each isolate and treatment) were incubated under high- and reduced-nutrient conditions for a week. High-nutrient treatments were full strength f/2+Si (i.e., the maintenance media), while low-nutrient treatments were a 10-fold dilution of f/2 + Si culture media with filter-sterilized seawater. The low-nutrient conditions were previously found to elicit increases in ingestion by these two species.

Ross Sea Dinoflagellate and Phaeocystis antarctica RNA-Seq

The Ross Sea Dinoflagellate (RSD) was enriched away from the prey and grown in f/2 + Si at 0C under 14/10hr light dark cycle at 26.2 µmol/m2/sec. For the temperature experiment, flasks with approximately 450,000 cells each incubated at 0C (4 replicates) or 5C (4 replicates) for 5 days. For the different light conditions, flasks of approximately 450,000 cells each were incubated under constant illumination of 371 µmol/m2/sec (surface; 4 replicates), 48 µmol/m2/sec (DCM; 4 replicates) or darkness (4 replicates) at 0C for 5 days.

Phaeocystis antarctica was grown under the same experimental conditions with 4 replicates per treatment. Approximately 3.3 x 10^6 cells per replicate were used in the 0C incubation, 2.6 x 10^6 cells per replicate in the 5C incubation, and 1.4 x 10^7 cells per replicate in the different light exposure treatments.

Sampling and analytical procedures:

Pyramimonas and Micromonas were collected on 25mm 0.2 µm polycarbonate filters, frozen at -80°C, then extracted using the Qiagen Mini RNA kit, with confirmation of nucleic acid abundance and quality assessed by Bioanalyzer. Replicate samples were sent to the Sulzberger Genome Center at Columbia University for poly-A selection, library construction and sequencing by Illumina Mi-Seq (150bp paired-end reads, 30 million reads per sample).

RSD and Phaeocystis cultures were fixed with RNALater and harvested by filtration onto 0.8 micron pore size 25 mm polycarbonate filters. Cells were lysed using the Qiagen RNeasy Mini Kit. DNA was removed by DNase treatment on the purification column. mRNA was isolated using ribosomal depletion (Ribo-Zero™ rRNA Removal Kit for Plant Leaf). RNA quality was checked by Bioanalyzer and sent to Sulzberger Columbia Genome Center for library preparation and RNA-Seq (150bp paired-end reads, 30 million reads per sample).