Unselected Metatranscriptome from 30Jul15 2am
Total RNA was extracted from filters using a Qiagen RNeasy Mini Kit modifying the lysis step with the addition of Biospec zirconia/silica beads. Synthetic standards (Ambion ERCC RNA Spike-in mix) were then added based upon estimated total RNA (using extraction values from prior samples collected in this region, Alexander et al. 2015, PNAS). The rest of the Qiagen RNeasy Mini Kit protocol was then followed according to the manufacturer's instructions, incorporating the on-column DNase digestion step, using a Qiagen RNase-free DNase kit. Resulting total RNA was eluted with RNase-free water and then purified and concentrated with an RNeasy MinElute kit according to the manufacturer s instructions. Extracted samples were split into two equal volumes where one volume was sequenced using the polyA pull-down step (Illumina Truseq library preparation kit) to enrich for eukaryotic mRNA (referred to as  selected ) and one volume was sequenced skipping the polyA pull-down step (referred to as  unselected ). Libraries were sequenced with an Illumina HiSeq 2000 at the Columbia Genome Center. Samples targeting eukaryotic signals (selected) were sequenced to produce 90 million 100-bp, paired-end reads while samples targeting prokaryotic signals (unselected) were sequenced to produce 60 million 100-bp, paired-end reads.