This dataset provides the code-base for RNAseq data pipelining and analysis as part of the U.S. EPA, Great Lakes Toxicology and Ecology Division's ecological high throughput transcriptomics research program. The scripts are designed for use in either the U.S. EPA High Performance Computing (HPC) system or in R/R-studio. The scripts cover the entire workflow, starting with raw FASTQ files, to data preprocessing and quality control to differential gene expression analysis, benchmark dose modeling, and functional enrichment analysis. The code is accompanied by a protocol designed to be accessible to researchers with no prior experience in coding or working within a Linux-based computing environment. It aims to provide detailed, step-by-step instructions that will enable users to navigate through the various stages of the analysis pipeline effectively.