title: 5um size, eukaryotic fraction from incubation started on sample date of HoeDylan_S2 (24 August 2012) and run for 168 hours; no addition
design: Seawater was collected at the HOT station ALOHA (22˚45’ N, 158˚ W) from 25 m depth at 14:00 local time six times during August and September 2012 as part of the HOE-DYLAN sampling initiative aboard the R/V Kilo Moana. Hydrocasts for sampling were performed using a conductivity-temperature-depth (CTD) rosette water sampler equipped with 24 Scripps 12-L sampling bottles. Water was collected in acid-washed 20-L carboys and approximately 60 L of seawater was prescreened through 200 µm mesh and then filtered onto polycarbonate filters (5.0 µm pore size, 47 mm, Whatman) by way of peristaltic pump. Filters were changed every 20 minutes or when flow rate decreased. Filters were placed in cryovials and stored in liquid nitrogen until mRNA extraction. Total filter time was never more than 3 hours. An incubation experiment was performed with six treatments: DSW addition, +N, +P, -N, -P, and control no addition. Triplicate 20-L carboys of each treatment were incubated at ~30% surface PAR in on-deck incubators for seven days and harvested as described above, on the seventh day at 14:00 local.         RNA was extracted from individual filters with the mRNAeasy Mini Kit (Qiagen), following a modified version of the yeast protocol. Briefly, lysis buffer and mRNA-clean zircon beads was added to the filter and samples were vortexed for 1 minute, placed on ice for 30 seconds, and then vortexed again for 1 minute. Samples were then processed following the yeast protocol. The resulting RNA was eluted in water and then treated for possible DNA contamination using TURBO DNA-free Kit (Ambion) following the Rigorous DNase protocol. RNA from individual filters was then pooled by sample, using the RNA Cleanup Protocol from the mRNAeasy Mini Kit (Qiagen). The resulting mRNA sample thus represented approximately 60 L of total seawater for the in situ sample. Filters were pooled across like triplicate bottles by treatment, totaling 60 L from each of the incubation treatments. The total RNA sample was then enriched for eukaryotic mRNA through a poly-A pull down. The resulting enriched mRNA sample then went through library preparation with the Illumina TruSeq mRNA Prep Kit (Illumina). Libraries were sequenced with the Illumina HiSeq2000 at Columbia Genome Center. Each sample was sequenced to produce a targeted 60 million, 100 base pair, paired end. Raw sequence data quality was visualized using FastQC and then cleaned and trimmed using Trimmomatic v 0.27 (paired end mode; 6-base pair wide sliding window for quality below 20; minimum length 25 base pair).