454 sequencing of Taxus cuspidata transcriptome
Approximately 2.5 microgramme of poly (A)  RNA was isolated using Oligotex mRNA Midi Kit (Qiagen, Hilden, Germany) from the needles of T. cuspidata. cDNA was synthesized using the SMART PCR cDNA Synthesis Kit (Clontech, Mountain View, CA, USA). The double-strand cDNA was amplified using PCR Advantage II polymerase and the following thermal profile was applied: 1 min at 95 degree celsius followed by 13 cycles of 95 degree celsius for 15 sec, then at 65 degree celsius for 30 sec, the last at 68 degree celsius for 6 minutes (Clontech, Mountain View, CA, USA). The amplified cDNA product was purified with the PureLink PCR Purification Kit (Invitrogen, Carlsbad, CA, USA) using Buffer HC to remove fragments less than 300 bp.