Ion torrent kits

Total RNA was isolated from the VOR in vitro q24h multidose simulation study cell pellets (described above) using the RNeasy minikit (Qiagen, Valencia, CA) according to the manufacturer’s protocol with modifications described above. RNA samples were quantitated using the Qubit RNA high-sensitivity (HS) assay kit and the Qubit 3.0 fluorometer (Life Technologies, Carlsbad, CA). RNA (30 ng) was converted to cDNA using the SuperScript Vilo kit (Life Technologies, Carlsbad, CA) per the manufacturer’s protocol. Library barcoding, preparation, and sequencing were carried out per the manufacturer’s protocols using the following Ion Torrent kits on the Ion Chef Prep station and the Ion Torrent S5-XL (Life Technologies, Carlsbad, CA): Ion AmpliSeq transcriptome human gene expression panel Chef-ready kit (catalog no. A31446), Ion 540 chip kit (catalog no. A27766) and Ion 540 Kit-Chef (catalog no. A30011) (Life Technologies, Carlsbad, CA). Eight samples per 540 chip were sequenced with 8 to 10 million reads/sample. Sequencing from the AmpliSeq analysis was analyzed using Partek Flow software to evaluate genes modulated with RPM of >70, an FDR of <0.1, and a VOR/DMSO ratio of >1.5 or <0.75 for each time point.

All procedures for emulsion PCR and next-generation sequencing were performed with Ion Torrent equipment and Ion Torrent kits under the manufacturer specifications (Life Technologies, Carlsbad, CA, U.S.A.): emulsion PCR was performed with the Ion OneTouch 200 template kit in an Ion OneTouch. Enrichment of template positive Ionospheres (ISPs) was performed with an Ion OneTouch ES (Life Technologies, Carlsbad, CA, U.S.A.). Sequencing of enriched templates bound to ionospheres was done using the Ion PGM 200 sequencing kit in an Ion PGM Sequencer with either 314 or 316 chips. FASTQ files of each barcoded group of sequences were recovered from the Ion Torrent server for further analysis. Reads have been deposited in the Sequence Read Archive (SRA) from NCBI under study accession number: SRP052626.