SYSTCO II - Foraminifera in 42 deep-sea sediments nested subsamples of the Southern Ocean deep-sea sediment
For each of the 42 samples, we selected one out of the two positive PCRs obtained for library-preparation and Illumina sequencing. First, we purified a total of 10 PCR products using the High Pure PCR Cleanup Micro Kit (Roche) and we quantified them using the fluorometric quantitation method based on the Qubit® dsDNA HS Assay Kit (Invitrogen). We then run an electrophoresis migration of the 10 absolutely quantified products along with the other 32 PCR products. We quantified the 32 PCR products relatively using band intensities extracted by the ImageLab 4.0.1 software on the Gel DocTM XR+ transilluminator (BioRad). Approximately 10 ng of amplicons were then pooled for each sample. We then purified the pool of PCR products as above (High Pure PCR Cleanup Micro Kit, Roche) in order to discard primer dimerization products in three batches of 100 μl. Finally, we eluted each of the three fractions of amplicons in 10 μl, pooled them together, and quantified the pool using the same fluorometric method. We sent 28 μl of DNA at a concentration of 34.7 ng μl-1 for library-preparation and sequencing at Fasteris SA (Plan-les-Ouates, Switzerland). The library was prepared using the TruSeq genomic DNA reagents, starting with a A-tailing step, a ligation step including 0.5 μl of polynucleotide kinase and 1 μl of ATP 1 mM in addition to the adapter and ligation mix, and after purification, 8 PCR cycles for library enrichment according to Illumina instructions.