S1 mRNA-enriched cDNA shotgun library
Nucleic acids (DNA and RNA) were simultaneously extracted from each of the two samples using a phenol:chloroform protocol and mechanical shearing (Urich et al., 2008). To account for differences in extraction efficiencies between easily- and difficultly lysed cells, bead-beating was performed at two different speeds in the Fast-Prep instrument (4 and 5 m/s). The triplicate extractions at both conditions were later on merged into one nucleic acid pool per sample. After DNase treatment and RNA purification using MegaClear columns (Ambion), the success of the DNase treatment was verified by PCR with 16S rRNA primers specific for bacteria and archaea. Subsequently, total RNA was subjected to double-stranded cDNA synthesis as previously described (Urich et al., 2008). Shotgun sequencing of the HMW DNA and the two cDNA preparations was performed on a GS FLX pyrosequencer (Margulies et al., 2005) using Titanium chemistry at the Norwegian High-throughput Sequencing Centre located at the University of Oslo, Norway. All sequencing reads shorter than 150 bp were removed as well as those containing degenerate bases (‘Ns’) or an average quality score below 25