High throughput transcriptome sequencing of Hevea brasiliensis by 454 pyrosequencing 
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Total RNA was extracted using Concert??? Plant RNA Reagent (Invitrogen).  200 ng of mRNA samples were isolated using Absolutely mRNA Purification kit (Stratagene) and fragmented in 10x Fragmentation Buffer (0.1 M ZnCl2, 0.1 M Tris-HCl, pH7.0) at 70??C for 30 sec.  The reaction was stopped by adding 2 ??l 0.5 M EDTA and 28 ??l 10 mM Tris HCl, pH7.5.  The mRNA samples were cleaned using Agencourt RNAClean reagent (Beckman Coulter) and eluted in 19 ??l 10 mM Tris HCl, pH7.5.  Fragmented mRNA samples were converted to double strand cDNA according to the cDNA Synthesis System Kit (Roche) using random primers.  cDNA libraries for 454 pyrosequencing were prepared according to the cDNA Rapid Library Preparation (Roche) and amplified in emulsion PCR and subject to pyrosequencing using the GS FLX platform.  