RNA-Seq Protocol for Differential Gene Expression

Basically, the analysis was performed using methods previously reported by our group^{23 (link)}. RNA libraries were generated using an Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific) according to the manufacturer’s instructions. Emulsion PCR was carried out with an Ion OneTouchTM system and an Ion OneTouch 200 Template Kit v3 (Thermo Fisher Scientific). Template-positive Ion Sphere™ particles were enriched and purified for the sequencing reaction with an Ion OneTouch™ ES system (Thermo Fisher Scientific). The template-positive Ion Sphere™ Particles were loaded onto Ion PI™ Chips (Thermo Fisher Scientific) and for high throughput sequencing with an Ion Proton™ Semiconductor sequencer (Thermo Fisher Scientific). Sequencing data were mapped on a human reference genome sequence (GRCh38/hg38) using the Torrent Suite software program (Life Technologies). The expression analysis was performed in the CLC Genomics Workbench software program (CLC bio, Aarhus, Denmark), and differences among the samples were determined using an unpaired Student’s t-test. The gene list describing fold change and p-value was uploaded to the MetaCore software (Clarivate Analytics, PA, USA, URL; https://portal.genego.com/, version 6.33.69110.), and then pathway analysis was performed.