GSM7864574: TW_Cisplatin_TERT_OAT1_Apical_3; Homo sapiens; RNA-Seq
Cell lysates were used for the Templated Oligonucleotide Sequencing Assay (TempO-Seq™, BioSpyder Technologies) assay as mRNA quantitation technology of choice (House et al., 2017). Detailed protocols for TempO-seq are provided by the manufacturer and were previously reported detailed elsewhere (House et al., 2022). TempO-seq libraries were prepared using the human S1500+ targeted transcriptome panel (Mav et al., 2018) consisting of 2,982 transcripts according to the manufacturer's instructions. Briefly, hybridization of the mRNA content of cell lysates was achieved by incubation of 2 μL of the lysate with 2 μL hybridization mix as follows: 10 min at 70°C, cooling ramp from 70°C to 45°C for 49 min, and 1 min at 45°C. Excess oligonucleotides were then digested in a nuclease catalyzed reaction for 90 min at 37°C. Next, hybridization products were incubated with DNA ligase for 60 min at 37°C. Nuclease and ligase were then denatured using a heat denaturation step (80°C for 30 min). A total of 10 μl of each ligation product were then mixed with an equal volume of PCR amplification mix and amplified in a LightCycler 96 (Roche, Basel, Switzerland) using the manufacturer recommended settings. Amplicon samples (5 μl) were then pooled and purified using a commercial PCR clean-up kit (Clontech, Mountain View, CA). Pooled libraries were sequenced in 75 single-end read mode dual index run with custom primers using NextSeq 550 sequencing (Illumina, San Diego, CA).