BioSample

Protocols: Leaves from 4 week-old plants were harvested 24 hours after whole-plant infection with Phytopthora infestans RNA was isolated by RNeasy Plant Mini Kit (Qiagen) Beads with oligo(dT) were used to isolate poly(A) mRNA. Fragmentation buffer was added for interrupting mRNA to short fragments. Taking these short fragments as templates, Random hexamerprimer were used to synthesize the first-strand cDNA. The second-strand cDNA was synthesized using buffer, dNTPs, RNase H and DNA polymerase I, respectively. Short fragments were purified with QiaQuick PCR extraction kit and resolved with EB buffer for end reparation and adding poly(A). After that, the short fragments were connected with sequencing adaptors. And, for amplification with PCR, we selected suitable fragments, as templates, with respect to the result of agarose gel electrophoresis.