RNA-Seq of PaWB phytoplasma-infected Paulownia witches
Total RNA was extracted from 3g of central leaf midribs and petioles from diseased and healthy paulownia using a TRIzol® Reagent (Invotrigen) following the manufacturer’s instructions, and then treated with DNase I (Invitrogen). RNA concentration and integrity were analyzed by Agilent 2100 Bioanalyzer (Agilent Technologies). Equal quantities of total RNA from four samples were mixed. Poly(A)-containing mRNA was isolated using magnetic beads with oligo(dT) and interrupted into short fragments. These short fragments were used as templates to synthesize the first-strand cDNA using reverse transcriptase and random hexamer-primers. And then the second-strand cDNA was synthesized using DNA polymerase I, dNTPs and RNase H. The double-strand cDNA fragments were purified, end repaired, added with poly (A) and finally connected with sequencing adapters. After the agarose gel electrophoresis, the suitable fragments were amplified through PCR to obtain the final library.