Castanea sativa and Castanea crenata Transcriptome and Gene Expression
Poly(A) RNA was enriched from  15-30 µg of total RNA using the MicroPoly(A) Purist Kit (Applied Biosystems, Ambion, CA) according the manufacturer’s instructions, followed by a second round of oligo (dT) selection.  The purity of  Poly(A)+ RNA was verified on Agilent 2100 Bioanalyzer with the RNA 6000 Pico kit and the quantity assessed by fluorometry with the Quant-iT RiboGreen RNA kit.  A fraction of 200 nanograms of Poly(A)+ RNA of each isolate was used as starting material for cDNA library construction using Multiplex Identifiers (MIDs) according the cDNA Rapid Library Preparation Method Manual, GS FLX Titanium Series, October 2009 (Roche-454 Life Sciences, Brandford, CT, USA). Briefly, preparing the cDNA libraries consisted of fragmentation of RNA, double-stranded cDNA synthesis using the cDNA Synthesis System Kit with Primer ‘‘random’’ (Roche Diagnostics GmbH, Mannheim, Germany), rapid library MID adaptor ligation and cDNA library quality assessment with a High Sensitivity DNA chip on Agilent 2100 Bioanalyzer.   The four dsDNA libraries were quantified by fluorescence, pooled in equimolar amounts and pyrosequenced at Biocant (Biocant, Cantanhede, Portugal) in a single plate with GS FLX Titanium chemistry (Roche-454 Life Sciences, Brandford, CT, USA),  according the standard manufacturers’ procedures.
BioFIG
Castanea crenata inoculated