BioSample

Protocols: Burkholderia phytofirmans PsJN colonizing potato (Solanum tuberosum L.) plants From the rRNA depleted RNA samples, plant mRNA molecules were removed with oligo(dT) magnetic beads. The poly(A)-minus RNA species were poly(A)-tailed using poly(A) polymerase and the RNA species which carry a 5' mono-phosphate were degraded with Terminator exonuclease (Epicentre). The samples were then treated with RNA 5' polyphosphatase (Epicentre) to degrade 5'PPP ends to 5'P. Afterwards, an RNA adapter was ligated to the 5'-phosphate of the RNA fragments. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and M-MLV reverse transcriptase. The resulting cDNA was PCR-amplified to about 20-30 ng/μl using a high fidelity DNA polymerase . The barcode sequences which are part of the 3' sequencing adapter are included in Table 2. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and analyzed by capillary electrophoresis.