Alexandrium CCMP 1719 transcriptome Experiment
The library was constructed by: Vertis Biotechnologie AG (http://www.vertis-biotech.com/).  In short, from the total RNA, polyA-RNA was prepared. First-strand cDNA synthesis was primed with a N6 randomized primer. Then 454 adapters A (CCATCTCATCCCTGCGTGTCTCCGACTCAG) and B (CTGAGACTGCCAAGGCACACAGGGGATAGG) were ligated to the 5'and 3' ends of the cDNA. The cDNAs were amplified with PCR (17 cycles) using a proof reading enzyme. Normalization was carried out by one cycle of denaturation and reassociation of the cDNAs, resulting in N1-cDNA. Reassociated ds-cDNA was separated from the remaining ss-cDNA (normalized cDNA) by passing the mixture over a hydroxylapatite column. After hydroxylapatite chromatography, the sscDNAs were amplified with 9 PCR cycles. For Titanium sequencing the cDNAs in the size range of 500 – 700 bp were eluted from a preparative agarose gel. The final cDNAs were double stranded, contained a barcode (CACACG), followed by the 5'Adapter (GACCTTGGCTGTCACTC),  the EST sequence, and finally the 3'Adapter (TCGCAGTGAGTGACAGGCCA).