A protocol for single nucleus RNA-seq from frozen skeletal muscle

Abstract

Single-cell technologies are a method of choice to obtain vast amounts of cell-specific transcriptional information under physiological and diseased states. Myogenic cells are resistant to single-cell RNA sequencing because of their large, multinucleated nature. Here, we report a novel, reliable, and cost-effective method to analyze frozen human skeletal muscle by single-nucleus RNA sequencing. This method yields all expected cell types for human skeletal muscle and works on tissue frozen for long periods of time and with significant pathological changes. Our method is ideal for studying banked samples with the intention of studying human muscle disease.

Identifying genetic variants that influence the abundance of cell states in single-cell data

“…Furthermore, high-dimensional profiling technologies can readily be applied to tissues that are difficult to disaggregate or lack a broad repertoire of markers for flow-sorting, as illustrated by single-nuclear profiling of brain and muscle samples and of frozen biobanked tissue. [32][33][34] However, these high-dimensional single-cell datasets present new statistical challenges. Instead of modeling genetic associations to a one-dimensional dependent variable (e.g., expression of one gene or abundance of one predefined cell type), analysis of these datasets requires a flexible approach capable of detecting genetic associations to many cell states, each defined by different combinations of the profiled cell features.…”