RNA-Seq of Argyranthemum frutescens
Total RNA was used as input material for the RNA sample preparations. Briefly, mRNA with polyA tail was enriched from total RNA using Oligo(dT) attached magnetic beads . Fragmentation was carried out using divalent cations under elevated temperature in Fragmentation Buffer . First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase , then use RNaseH to degrade the RNA. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and dNTPs. Remaining overhangs were converted into blunt ends via exonuclease / polymerase activities. After adenylation of 3 ends of DNA fragments. Adaptor with hairpin loop structure were ligated to prepare for hybridization. To select cDNA fragments of preferentially 370~424 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter , Beverly , USA). Then PCR was performed with Phusion High-Fidelity DNA polymerase , Universal PCR primers and Index (X) Primer.
SUB15154152
AF5