Dataset: Transcription in marine bacteria

Sampling and Analytical Methodology 

Bacterial strains. Pelagibacter ubique strain HTCC1062 and Gammaproteobacteria SAR92 strain HTCC2207 were obtained from S. Giovannoni (Oregon State University). Polaribacter strain MED152 was obtained from J. Pinhassi (Linnaeus University, Sweden). Ruegeria pomeroyi strain DSS-3 was obtained from Mary Ann Moran (University of Georgia).

Growth media and conditions. Pelagibacter and SAR92 were grown in a defined medium containing artificial seawater salts AMS1 (Carini et al. 2013) with additions of pyruvate, glycine, methionine and other organic substrates plus vitamins as described by (Steindler et al. 2011). Ruegeria was grown using YTSS medium that includes 0.4 g L-1 yeast extract and 0.25 g L-1 tryptone added to artificial seawater salts (Sigma-Aldrich). MED152 was grown in a medium with 5 g L-1 of peptone and 1 g L-1 of yeast extract added to the AMS1 salt solution. Cultures were maintained at 19°C in the dark and were aerated by bubbling with 0.2 µm filtered air or by rotary shaking.

Growth rates. Bacterial abundance was monitored by flow cytometry using a BD FACSCalibur of samples stained with SYBR Green I (Invitrogen) at a concentration of 1:2,000 of the manufactures supplied concentration for 30 min. Growth rates were calculated from the rate of change of bacterial abundance over time.

Nucleic acid extraction. Bacterial biomass was collected by vacuum filtration using 0.2 µm-pore-size Durapore (Millipore) filters. The filters were stored at -80°C in RLT buffer until DNA and RNA were extracted using the AllPrep DNA/RNA (Qiagen) kit following the manufacturer's instructions.

RNA sequencing abundance standard. Internal standard RNA molecules were used to obtain absolute quantification of transcripts based on the number of standard molecules added at the beginning of sample processing and those recovered in the sequence library (Moran et al. 2013, Satinsky et al. 2013). RNA standards were prepared using in vitro transcription (RiboMax Large Scale RNA Production Systems, Promega) from plasmid templates pTXB1 (New England BioLabs) and pFN18K (Promega), yielding single stranded RNA transcripts of 917 nt and 970 nt, respectively.   The RNA standards were added immediately before nucleic acid extraction at a concentration of 0.5% by mass of the total RNA yield in the sample (Moran et al. 2013, Satinsky et al. 2013).