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Supplementary methods
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Sample collection and labeling
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Lake Dagow is a small eutrophic lake in northern Germany with a high methanogenesis and
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methane oxidation potential. Samples were collected on 28 October 2010 from littoral
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sediment using a gravity corer as described previously (Dumont et al. 2011). The overlying
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water was oxic on the day of sampling, which would have enabled aerobic methane
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oxidation in the surface sediment in situ. 10 ml of the top 1 cm sediment was placed in 120
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ml serum bottles capped with butyl stoppers. An atmosphere containing 10% 13CH4 in air
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(Sigma Isotec, Taufkirchen, Germany) was provided and bottles were incubated in the dark
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at 4°C with gentle shaking. A total of 70 mol 13CH4 per ml of sediment was consumed after
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incubation for eight days. Total RNA was isolated as described previously (Dumont et al.
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2011) and stored at -80°C.
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Preparation of mRNA for sequencing
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The MICROBExpressTM system (Invitrogen, Karlsruhe, Germany) was used to enrich mRNA
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by removal of rRNA molecules, as described by the manufacturer. The enriched mRNA was
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subjected to isopycnic gradient centrifugation in CsTFA and gradient fractions with
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buoyant densities ~1.83 gml-1 and ~1.80 gml-1 were selected for downstream processing,
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as previously these fractions were shown to correspond to labelled and unlabelled RNA
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respectively (Dumont et al. 2011). RNA was quantifiied using the Quant iTTM RiboGreen kit
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(Invitrogen, Karlsruhe, Germany) and indicated that approximately 10 ng labelled and 300
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ng unlabelled RNA were obtained. To produce sufficient template for sequencing, RNA was
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amplified using the ExpressArt mRNA amplification kit, as described by the manufacturer
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(AmpTec, Hamburg, Germany). Totals of 20 and 27 g of amplified RNA (aRNA) were
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obtained from the heavy and light mRNA, respectively. To check for amplification bias, T-
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RFLP fingerprinting of pmoA transcripts was performed (Dumont et al. 2011) with the
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original RNA and aRNA and showed similar patterns, indicating minimal bias at least for
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pmoA mRNA (Fig. S1). The sizes of aRNA fragments were determined by electrophoresis
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using HighSens chips on an ExperionTM electrophoresis station (BioRad, Munich, Germany),
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and indicated a range of 0.2 to 2 kb with a median size of approximately 0.5 kb. Barcoded
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libraries were prepared according to the NEB Next Quick DNA Sample Prep Master Mix Set
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for 454 (NEB). Libraries were shotgun-sequenced on a Roche 454 GS FLX+ instrument with
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the GS FLX Titanium Sequencing Kit XLR70 and the Titanium PicoTiter-Plate Kit (Roche
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Diagnostics), following the manufacturer’s protocols.
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RNA sequencing and analysis
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The aRNA was converted to cDNA and sequenced at the Max Planck Genome Center
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(Cologne, Germany) using a Roche GS FLX+ system. A total of 103630 reads were obtained.
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Sequences shorter than 100 bp were removed from the dataset. Putative rRNA sequences
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were identified by BLASTN (version 2.2.25+) against a database containing the SILVA 108
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Parc databases of SSU and LSU rRNA sequences (Pruesse et al. 2007) using an expectation
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value threshold of 0.6 for saving hits, which empirically was found to discriminate well
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between mRNA and rRNA sequences. The remaining 15452 sequences were queried
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against the NCBI-nr database (17 April 2012) using TBLASTX (Altschul et al. 1997) and
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analyzed using MEGAN4 (Huson et al. 2011).
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References
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Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, et al. (1997) Gapped
BLAST and PSI-BLAST: a new generation of protein database search programs. Nuc Acid Res
25: 3389–3402.
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Dumont MG, Pommerenke B, Casper P, Conrad R. (2011) DNA-, rRNA- and mRNA-based
stable isotope probing of aerobic methanotrophs in lake sediment. Environ Microbiol 13:
1153–1167.
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Huson DH, Mitra S, Ruscheweyh H-J, Weber N, Schuster SC. (2011) Integrative analysis of
environmental sequences using MEGAN4. Genome Res 21: 1552–1560.
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Pruesse E, Quast C, Knittel K, Fuchs BM, Ludwig W, Peplies J, et al. (2007) SILVA: a
comprehensive online resource for quality checked and aligned ribosomal RNA sequence
data compatible with ARB. Nuc Acid Res 35: 7188–7196.
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