GSM7733634: Muscle Hindlimb, CON 56-days; Mus musculus; RNA-Seq
Briefly, the IR or the CON limb was removed at 24-hours post the final dose of radiation or 56-days post-IR (CON 24-hours, IR 24-hours, CON 56-days, and IR 56-days). Mechanical and enzymatic dissociation were used as previously described16 to generate a single-cell suspension from the IR or the CON hindlimb at the different time points. Briefly, the IR and the CON limb were dissected and processed separately. The connective and fat tissue was removed, and the dissected limb was subsequently transferred to a Miltenyi GentleMACS C tube (Cat# 130-093-237, Miltenyi Biotec), which contained an enzymatic solution comprised of Collagenase B (1.5U/ml, Cat# 11088831001; Roche® Life Science Products,) and Dispase II (2U/ml, Cat# 42613-33-2; Sigma Aldrich) resuspended in Ham's F10 media (Cat# 318-050-CL, Wisent Inc, St-Bruno, Canada). Using the gentleMACS Octo Dissociator (Cat# 130-096-427, Miltenyi Biotec), the muscle homogenate was passed through a 100 µm strainer (Cat# 22-363-549, Fisher Scientific). The resulting cell suspension was centrifuged at 600 x g at 4°C for 10 minutes. The cell pellet was treated with Red Blood Cell Lysing Buffer (Hydri-MaxTM; Cat# R7767; Sigma-Aldrich) and centrifuged at 600 x g at 4°C for 5 minutes. The cell pellet was then resuspended in 1 ml ice-cold FACS buffer, which consisted of 10% FBS and 3 mM EDTA in 1xPBS. For FACS, cell suspension from three limbs were pooled then were labelled with a live cell nuclei stain (NucBlue™ Live ReadyProbes™ Reagent (Hoechst 33342), Cat# R37605, Thermo Fisher Scientific), and SYTOX™ AADvanced™ (Cat# S10349, Thermo Fisher Scientific) for dead cells (Figure S7A). A minimum of 10,000 live, mononucleated cells were sorted in the Cell Sorting Facility at the Ottawa Hospital Research Institute using the Beckman Coulter MoFlo XDP cell sorter (32-bit high resolution digital system with five excitation laser lines (UV, 405nm, 488nm, 561nm, and 640nm) for further scRNA-seq. Library preparation were conducted by the manufacter's instructions (single cell 3', 10x Genomics)