Experimental design, methods, data processing, and results are further described in (Vorobev et al., 2018).
Acquisition Description:
Near-surface water samples were collected from coastal waters near Sapelo Island, Georgia (31° 25' 4.08" N, 81° 17' 43.26" W), ~6 km from the mouth of Doboy Sound during July 2014 and October 2014. Samples were collected in daylight between 7:30 and 18:30. Six 20 L carboys were filled with water and wrapped in black plastic. Three were processed immediately, while the remaining three were returned to Doboy Sound for a 24 h dark incubation before processing by an identical protocol. This experimental scheme was carried out twice during each sampling event, once at high tide and once at low tide. To process the samples, 3 L were passed through a 3 µm pore-size filter to remove eukaryotic cells (Capsule Pleated 3 µm Versapor Membrane; Pall Life Sciences, Ann Arbor, MI, USA) and then a 0.22 µm pore-size filter (Supor polyethersulfone; Pall Life Sciences). Subsamples for cell counts were collected prior to each filtration. The 0.22 µm filters were placed in Whirl-Pak® plastic bags (Nasco, Fort Artkinson, WI, USA) and immediately flash-frozen in liquid N2. The 0.22 µm filtrate was used for analysis of dissolved organic matter.
RNA was extracted from two replicate Time 0 h (T0) and Time 24 h (T24) filters (Table 1). The 50 ml lysis tubes contained 7.5 mL CTAB Extraction Solution (Teknova, Hollister, CA), 7.5 ml phenol:chloroform:isoamyl alcohol solution (25:24:1, pH 6.7), 750 µl 10% SDS, 200 µl 1% proteinase-K, 3 g RNA PowerSoil beads (Mo-Bio, Carlsbad, CA), and internal standards (described below). Frozen filters were broken into pieces inside the Whirl-Pak bags using a rubber mallet and transferred to the lysis tubes. Tubes were vortexed for 10 min, centrifuged, and the aqueous phase was transferred to a new tube. The extraction was performed again with chloroform, followed by centrifugation. RNA was precipitated from the aqueous phase with isopropanol, washed with cold 70% ethanol, and dissolved in 100 μl nuclease-free water. RNA was purified using an RNeasy Kit (Qiagen, Valencia, CA) followed by two successive treatments with the Turbo DNA-free kit (Invitrogen, Carlsbad, CA) to completely remove residual DNA.
Ribosomal RNA (rRNA) was selectively removed using biotinylated rRNA probes prepared for bacterial and archaeal 16S and 23S rRNA and eukaryotic 18S and 28S rRNA with DNA from a sample collected simultaneously (Stewart et al., 2010). Probe-bound rRNA was removed via hybridization to streptavidin-coated magnetic beads (New England Biolabs, Ipswich, MA), and successful removal of rRNA was confirmed using a 2200 TapeStation (Agilent Technologies, Santa Clara, CA).
rRNA-depleted samples were linearly amplified using the MessageAmp II-Bacteria Kit (Applied Biosystems, Austin, TX), and amplified mRNA was converted into cDNA using the Superscript III First Strand synthesis system (Invitrogen, Carlsbad, CA) with random primers, followed by the NEBnext mRNA second strand synthesis module (New England Biolabs,). Synthesized cDNA was purified using the PureLink PCR Micro Kit (Invitrogen) followed by ethanol precipitation and resuspension in 70 µL TE buffer, and stored at -80 degrees C until library preparation. The cDNA was sheared to ~300 bp with an E210 ultrasonicator (Covaris, Woburn, MA) and TruSeq libraries (Illumina Inc., San Diego, CA) were constructed. Libraries were sequenced on the Illumina HiSeq2500 platform to obtain 250 bp single end reads.
Internal Standard Addition - During sample processing, known copy numbers of two artificial internal mRNA standards ~1000 nt in length were added to each sample prior to cell lysis (Satinsky et al., 2014); and dx.doi.org/10.17504/protocols.io.ffwbjpe). Standards were synthesized using custom templates that were transcribed in vitro to RNA (Satinsky et al., 2013). The number of internal standards recovered in the sequence library was quantified via BLAST homology searches.
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