RNA-seq of Puccinia Polysora which causes southern corn rust
Poly(A) mRNA was enriched by Oligo (dT) magnetic beads. All the mRNA sequences were cut into short fragments by using fragmentation buffer. Taking the short fragments as templated, random hexamer primers were used to synthesize first-strand cDNA. Then the buffer, dNTPs, polymerase I and RNase H were added to synthesize second-strand cDNA. The cDNA fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, MA, USA), resolved with EB buffer for end reparation and A tailing, connected with sequencing adapters. PCR was performed using the Phusion High-Fidelity DNA polymerase, universal PCR primers and an index primer. PCR products were purified using AMPure XP system and assessed on the DNA high sensitivity chips using the Agilent Bioanalyzer 2100 system to construct the final cDNA library.
library_Ppolysora