Muscle stem cells were isolated by FACS using a triple-negative CD31−/CD45−/Sca1− and double-positive VCAM+/α7-integrin+ strategy as described previously (Chakkalakal et al., 2012 (link)). Antibody clones, sources and catalog numbers are described in supplementary Materials and Methods . Cells were seeded in sarcoma-derived extracellular matrix (ECM)-coated 96-well plates in rich growth medium [F10 (Gibco), 20% fetal bovine serum (FBS) (Gibco), (5 ng/ml) FGF2 (R&D Systems)] for behavior analysis. For single cell sequencing experiments, cells were seeded in sarcoma-derived ECM-coated 6-well plates and allowed to activate in plating media [Dulbecco's modified eagle media (DMEM), 10% horse serum] for 18 h prior to library preparation.
Cells for behavior analysis were seeded at 850 cells/well on sarcoma-derived ECM (Sigma-Aldrich) in 96-well plates. Cells were maintained in rich growth media [F10 (Gibco), 20% FBS (Gibco), (5 ng/ml) FGF2 (R&D Systems)]. For single cell sequencing experiments in which cells were activated, cells were seeded in sarcoma-derived ECM-coated 6-well plates and allowed to activate in plating media (DMEM, 10% horse serum) for 18 h prior to library preparation.
Cells for behavior analysis were seeded at 850 cells/well on sarcoma-derived ECM (Sigma-Aldrich) in 96-well plates. Cells were maintained in rich growth media [F10 (Gibco), 20% FBS (Gibco), (5 ng/ml) FGF2 (R&D Systems)]. For single cell sequencing experiments in which cells were activated, cells were seeded in sarcoma-derived ECM-coated 6-well plates and allowed to activate in plating media (DMEM, 10% horse serum) for 18 h prior to library preparation.
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