Ion pgm next generation sequencer

Genomic DNA was collected from peripheral blood samples of DMD patient (DMD02) and DMD gene was analyzed using Ion PGM next-generation sequencer (Thermo Fisher Scientific, Waltham, MA, United States).

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.