Elucidating molecular mechanisms and understanding genetic heterogeneity in drug discovery and complex biology can be achieved through high-throughput gene expression analysis and pathway assignment. Recent advances in RNA-seq methodologies have enabled accurate gene expression profiling from low-input and low-quality RNA samples, but analyzing hundreds or thousands of samples is an arduous exercise in library prep. QIAGEN’s QIAseq 3’ UPX technology provides a high-throughput RNA-seq library prep methodology where reverse transcription may be performed directly on lysed cells, while simultaneously assigning a unique ‘molecular index’ and ‘cell index’ to all cDNA synthesized from a sample. All subsequent full transcriptome or targeted panel library steps that follow occur in a single pooled tube. This technique allows ultra-plexing of thousands of samples per sequencing lane for a smart and resource-efficient workflow that enables the identification and characterization of gene signatures.