Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Total RNA extraction. Potato-inoculated and in vitro cultured bacterial cells were stabilized using the RNAprotect® reagent (Qiagen, USA) according to manufactures' instructions. Total RNA from in vitro grown and potato-inoculated bacteria was extracted as previously described using the RNeasy mini kit (Qiagen, Hilden, Germany) with slight modifications. The concentration and purity of each extracted total RNA sample was evaluated using spectrophotometric analysis (NanoDrop® ND-1000; NanoDrop® technologies, Wilmington, DE) at a ratio of 230/260 nm. Using the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.), total RNA samples' concentration, RIN and 28S/18S ratio were determined. cDNA library construction and sequencing. The Illumina sequencing service was provided by the BGI Co., Ltd (China). TruSeq RNA Sample Prep Kit v2 (Illumina, USA) was used to construct the cDNA libraries following the manufacturer's protocol. In summary, the mRNA was cleaved into small fragments, followed by the synthesis of first-strand cDNA with random hexamer-primed reverse transcription. RNase H and DNA polymerase I were used to synthesize second-strand cDNA. The double-stranded cDNA was subjected to terminal modification, by addition of adenosine and ligated with adapters. Adaptor-ligated fragments with suitable sizes were selected and enriched by PCR using the PureLinkTM PCR Purification Kit (Invitrogen, USA) to create the cDNA libraries for sequencing. The paired-end sequencing (PE91) was performed on the Illumina HiSeq 2000 sequencing platform.