title: Good vs bad quality cork: high-throughput sequence of phellogenic tissue
design: Biological sample  Phellogenic tissue was collected from 10 mature cork oaks under cork production (estimated age 50-70 years) in Southern Portugal. The sample was taken at the time of cork harvest, in the period of end July-beginning August 2011, by scratching the inside of the plank (the so-called cork belly, corresponding to the torn out phellogen) immediately after the separation of the cork plank from the tree stem, and freezing in liquid nitrogen right after the collection. Five samples were taken from cork oaks producing good quality cork and five samples from trees with bad quality cork were retained for analysis.   cDNA preparation  The quality of total RNA was verified on Agilent 2100 Bioanalyzer with the RNA 6000 Pico Kit (agilent Technologies, Waldbronn, Germany) and the quantity assessed by fluorimetry with the Quant-iT RiboGreen RNA kit (Invitrogen, CA, USA). A fraction of 0.5-2 micrograms of each pool of total RNA was used as starting material for cDNA synthesis with the MINT cDNA synthesis kit (Evrogen, Moscow, Russia), a strategy based on the SMART double-stranded cDNA synthesis methodology with amplification of polyA mRNA molecules using a modified template-switching approach that allows the introduction of known adapter sequences to both ends of the first strand cDNA. The synthesis were also done with a modified oligodT containing a restriction site for Bsgl to eliminate these tails and, in that way, minimize the interference of a A homopolymers during the 454 sequencing run.  Non-normalized cDNA was quantified by fluorescence and sequence in 454 GS FLX Titanium according to the standard manufacturer’s instructions (Roche-454 Life Sciences, Bradford, CT, USA) at Biocant (Cantanhede, Portugal).