Isopycnic centrifugation, fractionation and RNA-SIP library preparation

RNA from SIP experiments was extracted using the mirVana miRNA isolation kit (Ambion) with an added bead-beating step using RNA PowerSoil beads (MoBio, Carlsbad, CA, USA). A total volume of 100 μl was extracted and was then DNase treated using the Turbo-DNase kit (Ambion). Gradient preparation, isopycnic centrifugation, and gradient fractionation were performed as described in Lueders (2010). For each gradient sample, 5.1 ml of CsTFA (~2 g ml⁻¹, GE Healthcare Life Sciences, Piscataway, NJ, USA), 185 μl formamide, and a mixture of 750 ng RNA and gradient buffer solution (0.1 m Tris-HCl, 0.1 m KCl, 0.1 mM EDTA) up to 1 ml were first mixed in a 15 ml tube. Once mixed, the refractive index was measured for each sample to ensure a median density of ~1.80 g ml⁻¹. Samples were then loaded into 4.9 ml OptiSeal tubes (Beckman Coulter, Brea, CA, USA), placed into a VTi 65.2 vertical rotor (Beckman Coulter) and spun at 37 000 r.p.m. at 20 °C for 64 h using an Optima L-80 XP ultracentrifuge (Beckman Coulter). Each gradient was fractionated into 12 tubes of approximately 410 μl each and the refractory index of each fraction was measured to determine density. RNA was precipitated with isopropanol and the pellet was washed with 70% ethanol as described in Lueders (2010). RNA concentration of each fraction was determined using the RiboGreen quantification kit (Invitrogen) and a Gemini XPS plate reader (Molecular Devices, Sunnyvale, CA, USA).

Figure 1 depicts the density fractions chosen for sequencing. At each temperature, four metatranscriptomic libraries were constructed, two from the ¹²C-control, named ¹²C-light and ¹²C-heavy, and two from the ¹³C-experiment, named ¹³C-light and ¹³C-heavy. The two density fractions with the highest RNA concentration in the ¹²C-control incubation were combined to construct the ¹²C-light library, which represents the natural community. Similarly, two density fractions with the highest RNA concentration in the ¹³C-experiment incubation were combined to construct the ¹³C-heavy library, which represents the labeled autotrophic community. The other two libraries from each temperature, ¹²C-heavy and ¹³C-light, were constructed to act as additional controls. To construct the ¹²C-heavy library, density fractions were chosen that corresponded to the same density fractions used to construct the ¹³C-heavy library. The ¹²C-heavy library represents the heavier density unlabeled community and acts as a key comparison with the ¹³C-heavy labeled community to confirm that the populations in the ¹³C-heavy library have taken up the labeled isotope and are not naturally found at heavier nucleic acid densities. To construct the ¹³C-light library, density fractions were chosen that corresponded to the same density fractions used to construct the ¹²C-light library. The ¹³C-light library represents the lighter density community in the ¹³C-experiment, which includes both labeled and unlabeled populations that are found at lighter densities. For all libraries, double-stranded complementary DNA was constructed and metatranscriptomic library preparation and analysis was carried out as described in the Supplementary methods. Raw read data are available through the European Nucleotide Archive for all 20 RNA-SIP metatranscriptomes, under sample accession numbers ERS743595 through ERS743614.

Fractionation plots of RNA-SIP experiments for each of the three temperatures. Plots depict the RNA concentration (ng μl⁻¹) vs buoyant density (g ml⁻¹) for each fraction. RNA from fractions denoted by a symbol was used for metatranscriptomic library preparation and sequencing.