title: 454 sequencing of marine metagenome transcript fragment library
design:  For transcriptomics, total RNA was separated from the columns using the RNA MinEluteTM clean-up kit (Qiagen) and checked for integrity of rRNA using an Agilent bioanalyser (RNA nano6000 chip). High integrity rRNA is essential for subtractive hybridization. Samples were treated with Turbo DNA-free enzyme (Ambion) to remove contaminating DNA. The rRNA was removed from mRNA by subtractive hybridization (Microbe Express Kit, Ambion), and absence of rRNA and DNA contamination was confirmed using the Agilent bioanalyser. The mRNA was further purified with the MEGAclearTM kit (Ambion). Reverse transcription of mRNA was performed using the SuperScript Circle R III enzyme (Invitrogen) with random hexamer primers (Promega). The cDNA was treated with RiboShredderTM RNase Blend (Epicentre) to remove trace RNA contaminants. To improve the yield of cDNA, samples were subjected to random amplification using the GenomiPhi V2 method (GE Healthcare). GenomiPhi technology produces branched DNA molecules that are recalcitrant to the pyrosequencing methodology. Therefore amplified samples were treated with S1 nuclease using the method of Zhang et al.2006. 