Embryo transcriptome
Embryo transcriptome sequencing of Paris polyphylla var. yunnanensis
Approximately, 2ug of poly (A) RNA was isolated from equal mixtures of the three total RNA using an Oligotex m RNA Midi Kit (QIAGEN, CA). The long poly(A/T) tails in cDNA may lead to low-quality sequencing reads from the GS FLX system. To overcome this limitation, we designed a modified poly(T) primer with a BsgI site between the adaptor and the poly(T) 5'-AAGCAGTGGTATCAACGCAGAGTACT(20)VN-3') (Sun et al. 2010) . For cDNA synthesis, this poly(T) primer was used in combination with the Clontech SMART IV primer. The cDNA was then treated overnight with BsgI (NEB, MA, USA) at a concentration of 5 units/µg of cDNA. This restriction enzyme cut within the poly(A) tail, and greatly increased the quantity and quality of the sequencing reads. For the library, digested cDNA was amplified using PCR Advantage II polymerase ( Clontech,USA) and the following thermal profiles were used: 1min at 95°C followed by 16 cycles of 95°C for 15s, 65°C for 30s, and 68°C for 6min. PCR products(5ul) were determined by electrophoresis. Approximately 3ug ds cDNA was sent to the Beijing Institute of Genomics of the Chinese Academy of Sciences (Beijing ,China) for pyrosequencing using the 454-GS FLX Titanium Kit
Embryo cDNA
Institute of Medicinal Plants, Peking Union Medica
Embryo transcriptome