Transcriptomic analysis

Total RNA from rhesus macaque PBMC isolated using the TRIzol extraction method described above was prepared for transcriptomic analysis using the Illumina TruSeq Stranded mRNA Library Prep Kit (RS-122-2101, Illumina) as previously described [120]. The library was validated using an Agilent DNA 1000 kit on a bioanalyzer. Samples were sequenced by the OHSU Massively Parallel Sequencing Shared Resource using an Illumina NovaSeq.

Differential expression analysis was performed by the ONPRC Bioinformatics & Biostatistics Core. The quality of the raw sequencing files was evaluated using FastQC [121] combined with MultiQC [122] (http://multiqc.info/). Trimmomatic [123] was used to remove any remaining Illumina adapters. Reads were aligned to Ensembl’s Mmul_10 genome along with its corresponding annotation, release 109. The program STAR [124] (v2.7.10b_alpha_220111) was used to align the reads to the genome. STAR has been shown to perform well compared to other RNA-seq aligners [125]. Since STAR utilizes the gene annotation file, it also calculated the number of reads aligned to each gene. RNA-SeQC [126] and another round of MultiQC were utilized to ensure alignments were of sufficient quality.

Gene-level raw counts were filtered to remove genes with extremely low counts in many samples following the published guidelines [127], normalized using the trimmed mean of M-values method (TMM) [128], and transformed to log-counts per million with associated observational precision weights using the voom method [129]. Gene-wise linear models with primary variable day after infection, and accounting for within subject correlation, were employed for differential expression analyses using limma with empirical Bayes moderation [130] and false discovery rate (FDR) adjustment [131]. Differential expression data were analyzed through the use of IPA (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuity- pathway-analysis), using a stringent cutoff for significant molecules of FDRp < 0.2 and |FC| > 1.5. The background reference set used was the dataset of all genes in the differential analysis.