title: 5um size, eukaryotic fraction from incubation started on 2012-05-30 and run for 48 hours; Plus P treatment: Phosphate added
design: Filters from triplicate bottles, representing approximately 6 L of water, were pooled by treatment and extracted for each of the in situ and incubation experiment samples. RNA was extracted from individual filters with the RNAeasy Mini Kit (Qiagen), following a modified version of the yeast protocol. Briefly, lysis buffer and RNA-clean zircon beads were 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 each triplicate was then pooled by sample or treatment, using the RNA Cleanup Protocol from the RNAeasy Mini Kit (Qiagen). The total RNA ( > 1000 ng for each sample) was then enriched for eukaryotic mRNA through a poly-A pull down onto oligo-dT beads. The resulting enriched RNA sample then went through library preparation with the Illumina TruSeq RNA Prep Kit (Illumina). Libraries were sequenced at the Columbia University Genome Center (New York, New York) with an Illumina HiSeq2000. Each sample was sequenced to produce ~ 60 million, 100 base pair, paired end reads.