Extraction and sequencing of single nuclei from murine skeletal muscles

Abstract

Single-nucleus RNA sequencing allows the profiling of gene expression in isolated nuclei. Here, we describe a step-by-step protocol optimized for adult mouse skeletal muscles. This protocol provides two main advantages compared to the widely used single-cell protocol. First, it allows us to sequence the myonuclei of the multinucleated myofibers. Second, it circumvents the cell-dissociation-induced transcriptional modifications. For complete details on the use and execution of this protocol, please refer to Dos Santos et al. (2020) and Machado, Geara et al. (2021).

Keywords: Cell Differentiation; Cell isolation; Flow Cytometry/Mass Cytometry; RNAseq; Single Cell; Stem Cells.

Graphical abstract

Figure 1

Nuclei extraction from adult skeletal muscle (A) Amount of dissected skeletal muscle used for nuclei isolation. (B) The muscles are chopped with a pair of scissors for 2 min on ice. (C) Indicative picture of the muscles after chopping. The size of the muscle pieces should be around 1–2 mm. (D) Muscle preparation before and after Douncer homogenization. (E) Nuclei pellet after filtration and first centrifugation. A colorless pellet of the size shown in the picture is required.

Figure 2

Nuclear purification by DAPI-based cytometry (A) Nucleus preparations stained with DAPI after Douncer homogenization. Arrows point to debris (DAPI negative, solid arrows) and nuclei-debris aggregates (dotted arrow). Scale bar 400 μm. (B) Nuclei preparations stained with DAPI after FACS. An almost complete elimination of debris is evident. Scale bar 400 μm. (C) Nuclei from hindlimb and forelimb muscles of adult mice are sorted by FACS based on DAPI signal (≈50% DAPI+). Nuclei preparations stained with DAPI after FACS. Green arrows point to elongated nuclei, a characteristic feature of myonuclei, whereas gray arrows point at more rounded nuclei, probably deriving from other cell types. Scale bar 200 μm.

Figure 3

Expected results after sequencing and analysis by Cell Ranger and Seurat (A) Barcode rank plot showing the distribution of barcode counts and the barcodes associated with nuclei (from 10XGenomics). The curve must show a steep drop-off between the nuclei reads (left) and the background noise (right, degraded nuclei or empty partitions). If the nuclei preparation has too much contaminating ambient RNA, only one curve will be observed. The number of genes detected per nucleus must be between 700 and 2000. Y-axis: number of UMI counts mapped to each barcode; x-axis: number of barcodes. (B) Uniform Manifold Approximation and Projection (UMAP) diagram from snRNA-seq from adult skeletal muscle. MuSC: skeletal muscle stem cells, FAPs: fibro-adipogenic progenitors, MTJ: myotendinous junction, NMJ: neuromuscular junction. (C) Table of the different cell populations detected by snRNA-seq on skeletal muscle. The markers used to identify the different clusters are displayed in the middle and the percentage of nuclei for each population is displayed on the right.

Figure 4

Nuclear GFP signal is lost during nuclei purification but can be preserved with mild formaldehyde fixation (A) Forelimb and hindlimb muscles from 5.5-week-old Tg:Pax7-nGFP mice were manually dissected and chopped into small pieces and undergo nuclei purification with a Douncer homogenizer. For GFP preservation, samples are first fixed prior to homogenization in ice-cold 0.25% paraformaldehyde in PBS for 1 h at 4°C, then washed 3 × 10 min with PBS and are finally treated for FACS purification. (B) Nuclei from not fixed samples sorted by FACS based on DAPI signal (65.1% DAPI+). Note that the nuclear GFP signal is undetectable. (C) Nuclei from fixed samples sorted by FACS based on DAPI (55.7% DAPI+) and nuclear GFP signal (1.1% GFP+ of DAPI+). (D) Nuclear GFP signal in single-cell preparations from Tg:Pax7-nGFP muscles allows sorting of GFP+ cells without the need for fixation (2.1% GFP+ of cell population).