Comprehensive cellular analysis with single-nucleus RNA-seq of archived PAXgene whole blood samples

Single-cell analysis of the large repositories of preserved whole blood samples stored in PAXgene blood RNA tubes has not yet been available. To address this gap, two nuclei-isolation techniques for single-nucleus RNA sequencing were evaluated: mechanical separation (MS), using an Acrodisc filter, and cell lysis (CL). While both methods captured nuclei from all major immune cell types, CL resulted in up to two orders of magnitude higher nuclei yields and less biased proportions of immune cells than MS. High ambient globin gene counts following CL were substantially reduced by CRISPR-guided globin gene depletion of complementary DNA, resulting in more sensitive and efficient gene detection per cell. Despite capturing only nuclear transcripts, CL-derived samples maintained similar cell-type proportions and gene expression as matched peripheral blood mononuclear cell samples, while retaining granulocytes. The CL isolation with globin depletion method enables a comprehensive analysis of PAXgene whole blood samples at single-cell resolution.