Optimized Methods for scRNA-seq and snRNA-seq of Skeletal Muscle Stored in Nucleic Acid Stabilizing Preservative

Single cell studies have transformed our understanding of cellular heterogeneity in disease but the need for fresh starting material can be an obstacle, especially in the context of international multicenter studies and archived tissue. We developed a protocol to obtain high-quality cells and nuclei from dissected human skeletal muscle archived in the preservative Allprotect® Tissue Reagent. After fluorescent imaging microscopy confirmed intact nuclei, we performed four protocol variations that compared sequencing metrics between cells and nuclei enriched by either filtering or flow cytometry sorting. Cells and nuclei (either sorted or filtered) produced statistically identical transcriptional profiles and recapitulated 8 cell types present in skeletal muscle. Flow cytometry sorting successfully enriched for higher-quality cells and nuclei but resulted in an overall decrease in input material. Our protocol provides an important resource for obtaining high-quality single cell genomic material from archived tissue and to streamline global collaborative efforts. Overall design: Primary human skeletal muscle tissue from multiple donors was pooled, homogenized, filtered to create a single cell suspension, and divided into 4 aliquots. Aliquot1: unaltered. Aliquot2: nuclei extracted; Aliquot3: stained with Nuclear Pore Complex antibody (BioLegend #682202), and positively selected by fluorescence-activated cell sorting (FACS); Aliquot4: stained with Nuclear Pore Complex antibody, positively selected by FACS, and nuclei extracted.