Figure 1. Overview of high-throughput transcriptomics workflow.

(A) Diagram of initial plating of test and reference chemicals on a TempO-Seq dose plate, followed by randomization of chemical exposures to test plates. The first column of each test plate is not loaded with cells and is reserved for dispensing of QC samples. Grey wells on the test plate are reserved for internal positive controls added and verified by the contractor. (B) Diagram of bioinformatics workflow. Light yellow boxes indicate raw data received from contractor after targeted RNA-Seq assay completion. Light green circles indicate steps performed using existing open-source methods. Light blue circles indicate novel methods developed as part of this work. Bioinformatic analysis is generally split into two phases. Raw data processing up to probe-level count matrix and samplelevel QC metrics is performed across entire data set. Subsequent analysis is performed separately for each chemical against plate-matched vehicle controls. Intermediate processing results are stored in a database layer to facilitate analysis.