SEQURNA enhances FLASH-seq gene detection while eliminating DTT dependence

Abstract Effective RNase inhibition is critical for single-cell RNA-sequencing, yet commercial recombinant RNase inhibitors (RRIs) require reducing agents for stability and impose substantial costs. Here, we systematically benchmark SEQURNA, a synthetic thermostable RNase inhibitor, against commercial alternatives using FLASH-seq in human retinal organoids and peripheral blood mononuclear cells (PBMCs). SEQURNA at 0.5–0.75 U/μl achieved 12–15% higher gene detection than Takara RRI in both cell types (p < 0.001) . Surprisingly, DTT supplementation at standard concentrations (5–10 mM) significantly impaired gene detection across all SEQURNA concentrations without improving RNA quality metrics, challenging established reverse transcription protocols. SEQURNA preserved biological heterogeneity, maintained sample stability during one-month storage at − 80 °C, and reduced reagent costs by over 75%. We recommend SEQURNA at 0.75 U/μl without DTT as an optimized formulation that simultaneously enhances data quality and cost-effectiveness for full-length single-cell RNAsequencing.