RNA-Seq Library Preparation and Analysis

mRNA libraries were constructed using a TruSeq Stranded mRNA Library Prep kit V2 (Illumina) and were paired-end sequenced on a HiSeq 4000 system (Illumina) at the NCI Frederick Sequencing core facility.
RNA-Seq reads were processed using the Snakemake-based lcdb-wf transcriptomics pipeline v1.3c (https://github.com/lcdb/lcdb-wf) (52 (link)). Briefly, quality was assessed using FastQ Screen and FastQC, and adapter sequences and low-quality bps were trimmed using cutadapt. Trimmed reads were then mapped to the human reference genome (gencode v30) using HISAT2 (v2.1.0), and transcript levels were quantified using the featureCounts method from RSubRead (v1.6.4). Differential expression analysis was performed in the R/Bioconductor environment (R v4.0.2; Bioconductor v2.48.0) using the DESeq2 package (v1.28.0) (53 (link), 54 (link)). After constructing a DESeqDataSet object from the read counts generated by RSubRead, generalized linear models were fit with default parameters using a treatment coding design (e.g., NHWD870 versus vehicle). Finally, shrunken log-fold changes were estimated for each contrast using the apeglm adaptive t prior shrinkage approach (55 (link)).