TPD-seq: A high throughput RNA-seq method to derive transcriptomic points of departure from cell lines

There is growing scientific and regulatory interest in transcriptomic points of departure (tPOD) values from high-throughput in vitro experiments. To further help democratize tPOD research, here we outline 'TPD-seq' which links microplate-based exposure methods involving cell lines for human (Caco-2, Hep G2) and environmental (rainbow trout RTgill-W1) health, with a commercially available RNA-seq kit, with a cloud-based bioinformatics tool (ExpressAnalyst.ca). We applied the TPD-seq workflow to derive tPODs for solvents (dimethyl sulfoxide, DMSO; methanol) and positive controls (3,4-dichloroaniline, DCA; hydrogen peroxide, H₂O₂) commonly used in toxicity testing. The majority of reads mapped to protein coding genes (∼9 k for fish cells; ∼6 k for human cells), and about 50 % of differentially expressed genes were curve-fitted from which 90 % yielded gene benchmark doses. The most robust transcriptomic responses were caused by DMSO exposure, and tPOD values were 31-155 mM across the cell lines. OECD test guideline 249 (RTgill-W1 cells) recommends the use of DCA and here we calculated a tPOD of ∼5 to 76 μM. Finally, exposure of the two human cell lines to H₂O₂ resulted in tPOD values that ranged from 0.7 to 1.1 mM in Caco-2 cells and 5-30 μM in Hep G2 cells. The methods outlined here are designed to be performed in laboratories with basic molecular and cell culture facilities, and the performance and scalability of the TPD-seq workflow can be determined with additional case studies.