RNA-Seq and Gene Set Enrichment Analysis (GSEA) in Peripheral Blood Mononuclear Cells (PBMCs).

RNA sequencing (RNA-Seq) and analysis methods like gene set enrichment analysis (GSEA) stand at the forefront of modern molecular biology, offering unparalleled insights into gene expression dynamics and transcriptomic landscapes. In this chapter, we focus on the application of RNA-Seq technology and the GSEA method. First, we provide a real-world workflow for RNA-Seq to unravel the intricate interplay between host factors and HIV-1 infection within peripheral blood mononuclear cells (PBMCs). We elucidate the core principles of RNA-Seq and its pivotal role in identifying HIV-1 restriction factors and understanding their regulatory mechanisms in PBMCs. We provide the essential steps of the RNA-Seq workflow tailored to PBMCs, encompassing sample preparation, library construction, sequencing, and advanced bioinformatics. We underscore how RNA-Seq enables precise quantification of gene expression levels, detection of alternative splicing, and exploration of novel transcripts pertinent to HIV-1 restriction. Moreover, we delve into the intricacies of HIV-1 restriction factor discovery using RNA-Seq data, shedding light on the diverse repertoire of PBMC host defense mechanisms. We discuss the challenges of RNA-Seq data analysis, including normalization, differential expression analysis, and the integration of multi-omics datasets, and propose strategies to mitigate these barriers. Next, we provide an overview of GSEA, a powerful computational method that extends beyond traditional differential gene expression analysis. This tool can provide deeper insights into the functional significance of gene expression changes. By leveraging curated gene sets representing biological pathways, molecular functions, or cellular processes, GSEA enables the identification of coordinated changes in gene expression within predefined gene sets. In our example, we applied GSEA in transcriptomic studies, highlighting its ability to uncover subtle yet coordinated changes in gene expression that may not be evident by individual gene analysis. We demonstrate how GSEA enhances our understanding of complex biological phenomena, informs hypothesis generation, and identifies potential therapeutic targets. Overall, RNA-Seq and GSEA are indispensable tools for elucidating the complex interactions in host cells altered by physiologic processes, disease states or infectious agents like HIV-1, thereby advancing our ability to identify potential targets for therapeutic interventions.