Transcriptome profiles in Ziziphus jujuba Response to Jujube Witches’-Broom Phytoplasma Infection: healthy plant
Total RNA from a leave and a stem of healthy plant was extracted with the agent TRIzol (Invitrogen Co.). We used RNA-free DNase I (New England Biolab) to remove the residual genomic DNA. Equal amounts of the qualified RNA (OD260/OD280 = 1.6) of the two tissues was mixed in preparation for RNA-seq library construction. The library was constructed by the BGI Company (Shenzhen, China) following the steps. The mRNA was isolated using oligo(dT) beads and sheared into short fragments. Taking these short fragments as templates, random hexamer-primers were used to synthesize the first-strand cDNA. The second-strand cDNA was synthesized using a buffer, dNTPs, RNaseH and DNA polymerase I. Short fragments were purified using a QiaQuick PCR extraction kit, and resolved with EB buffer for end reparation and tailing A. After adapter ligation and agarose gel electrophoresis, suitable fragments were selected for PCR amplification. Finally, one RNA-seq library was sequenced using the Illumina HiSeq™ 2000.