High-throughput transcriptomics platform for screening hepatotoxicants

We introduce a new high-throughput transcriptomics (HTTr) platform comprised of a collagen sandwich primary rat hepatocyte culture and the TempO-Seq assay for screening and prioritizing potential hepatotoxicants. We selected 14 chemicals based on their risk of drug-induced liver injury (DILI) and tested them in hepatocytes at two treatment concentrations. HTTr data was generated using the TempO-Seq whole transcriptome and S1500+ assays. The HTTr platform exhibited high reproducibility between technical replicates (r>0.9) but biological replication was greater for TempO-Seq S1500+ (r>0.85) than for the whole transcriptome (r>0.7). Reproducibility between biological replicates was dependent on the strength of transcriptional effects induced by a chemical treatment. Despite targeting a smaller number of genes, the S1500+ assay clustered chemical treatments and produced gene set enrichment analysis (GSEA) scores comparable to those of the whole transcriptome. Connectivity mapping showed a high-level of reproducibility between TempO-Seq data and Affymetrix GeneChip data from the Open TG-GATES project with high concordance between the S1500+ gene set and whole transcriptome. Taken together, our results provide guidance on selecting the number of technical and biological replicates and support the use of TempO-Seq S1500+ assay for a high-throughput platform for screening hepatotoxicants. Overall design: Primary rat hepatocytes freshly isolated from male Wistar rats (250-300 g) were maintained in collagen sandwich in 96-well plates, and treated with 14 chemicals at two concentrations. To examine technical reproducibility, each treatment was performed in triplicate in culture medium containing 0.5 % DMSO for 24 h. To examine biological reproducibility, the entire experiment was repeated using hepatocytes isolated from another individual with library construction and sequencing independently performed from the first batch of samples.