Gs flx titanium series reagents

A minimum of 10 μg of DNA were used to generate metagenomic libraries for each Roche/454 pyrosequencing run on a 454 pyrosequencer (GS FLX Titanium Series Reagents; Roche 454; Shirley, NY, USA). In a first phase, pair-end sequencing was done on duplicate microcosms of each ESC (about one million reads per microcosm). As an exception, the three replicates of the mercury enrichment #2 were sequenced due to a low reproducibility of one microcosm observed using RISA profiles. Thus, 23 metagenomic data were generated. In a second phase, a mate-pair sequencing effort with 3 kb of gap was done for duplicates corresponding to 3 promising ESCs: heavy metals enrichment #2 (one million reads each), mercury enrichment #1 (two million reads each) and the outlier replicate of the mercury enrichment #2 (one million reads). Duplicates corresponding to the ethanol enrichment were also further sequenced, however, due to the high fragmentation of the DNA recovered from this ESC, additional sequencing was done with paired-end library preparation instead of mate-pair.

The cDNA library preparation and pyrosequencing were performed using GS-FLX Titanium series reagents following the manufacturer's instructions (Roche Diagnostics, Germany). An equal amount of mRNA (200 ng) from each strain was used to synthesize the first and second strands of the cDNA, according to the cDNA Rapid Library Preparation Method Manual GS FLX Titanium Series (Roche Diagnostics, Germany). After the library construction, the samples were quantified using the Qubit system (Invitrogen, USA), and average fragment sizes were determined using the Agilent 2100 Bioanalyzer (Agilent, USA). Three cDNA libraries for each strain were generated, totaling nine libraries, which were sequenced using the Roche/454 GS-FLX system. Following the analyses of the reads, each of the triplicate sets of libraries was pooled.

Total RNA was isolated from the samples using the NucleoSpin XS (Macherey-Nagel, Germany) following the manufacturer instructions. Immediately after RNA isolation, cDNA synthesis and amplification was performed using the SMARTer RACE cDNA amplification kit (TaKaRa BIO/Clontech, Mountain View, CA) followed by the library preparation protocol available on Matzlab website (http://www.bio.utexas.edu/research/matz_lab/). Approximately 2.5 μg of the cDNA pool from prepared libraries were used for a titration run using one-quarter of a plate for each sample on the Roche 454 Genome Sequencer FLX using GS-FLX Titanium series reagents. Sequencing was carried out at Genoscope Institute (Evry, France). Data have been submitted to the GenBank under the Sequence Read Archive (SRA) numbers SRR1734678 (A. elongata), SRR1744093 (Collozoum sp.), SRR1734679 (S. streptacantha), and SRR1734688 (A. scolymantha).