De novo transcriptome sequencing and comparative analysisof Cymbidium faberi Rolfe
Poly (A) mRNA was purified with oligo (dT) beads from total RNA, and then the mRNA-enriched RNA was randomly segmented into 200-700 nt fragments in a divalent cation fragmentation buffer (Illumina, Hayward, CA) for 8 min at 94°C. These short fragments were used as templates to synthesize the first-strand cDNA using random hexamer primers and the second-strand cDNA was generated using RNaseH and DNA polymerase I. Those short cDNA fragments were purified with QiaQuick PCR extraction kit and washed with elution buffer (EB) for end repairing and tailing-A. Then those short fragments were ligated to sequencing adapters according to Illumina’s protocol (San Diego, CA, USA) and further separated by agarose gel electrophoresis. Fragments of 300–500 bp were enriched by PCR amplification to create a cDNA library.