318 chip kit v2

The strains MAFF 212134 and MAFF 212141 (http://www.gene.affrc.go.jp/databases-micro_search_en.php) were picked up as the representative biovar 6 strains for genome sequencing. Their genomes were sequenced and assembled by using the same protocols used in our previous study^{5 (link)}, except that some reagents were different as follows; the Ion PGM Hi-Q View OT2 Kit (Thermo Fisher Scientific Inc.), the Ion PGM Hi-Q View Sequencing Kit (Thermo Fisher Scientific Inc.), and a 318 Chip Kit v2 (Thermo Fisher Scientific Inc.) were used in this study instead of the Ion PGM Template OT2 400 Kit, the Ion Sequencing 400 Kit, and a 318 Chip Kit, respectively. The assembled contigs of MAFF 212134 and MAFF 212141 were annotated using the NCBI PGAP (https://www.ncbi.nlm.nih.gov/genome/annotation_prok/), and registered in the nucleotide sequence databases (DDBJ/EMBL/GenBank).

Culture medium was prepared with or without 1000 ppm oxytetracycline. Four plates were prepared for each condition. For culture, 100 μL of inoculum was applied to each plate and then incubated for 2 weeks at 25°C. A culture sample was collected from each plate and used for RNA extraction with an RNeasy Plant Mini Kit (Qiagen). Following the manufacturer’s protocols, templates were created from sample RNAs with an Ion Total RNA-Seq kit v. 2 (Thermo Fisher Scientific Inc., Waltham, MA, United States) and an Ion PGM Hi-Q OT2 kit on an Ion OneTouch 2 system (Thermo Fisher Scientific). The templates were sequenced with an Ion PGM Hi-Q sequencing kit and a 318 Chip kit v. 2 on an Ion PGM next generation sequencer (Thermo Fisher Scientific). Sequence data were assembled and analyzed in CLC Genomic Workbench software (CLC Bio, Qiagen). The genome sequence of Ishi-1 (GenBank acc. no. AP014595) was used as the reference for RNA-Seq mapping and assembly of sequence reads. The gene expression value was calculated from RPKM (Reads Per Kilobase per Million mapped reads). Differential expression analysis used log₂(x + 0.0001) data. Principal coordinates analysis based on differential gene expression profiles was performed in the EdgeR package of R v. 3.3.0 software.