Single-Cell RNA Sequencing of GFP+ Cells and Skeletal Stem Cells in Cko Mice

For Sc-FACS-Seq analysis of GFP⁺ cells, BMCs were prepared from the hind limbs of P21 Cko mice (see above) and GFP⁺ cells were FACS sorted using a BD FACSAria II (Becton Dickinson) FACS sorter into a sterile 96-well plate at a density of one cell/well containing 10X lysis buffer (Clontech, # 635013) with Protector RNAse inhibitor (Roche). The plates were frozen immediately on dry-ice and stored at −80°C until ready to process for RNA-Seq. Two 96 well plates of GFP⁺ sorted single cells (GFP-Cko1, GFP-Cko2) were carried forward for analysis with each plate receiving cells sorted from a pool of BMCs made from two Cko mice. Thus, a total of four Cko mice were analyzed. For RNA-Seq analysis of skeletal stem cells (SSCs) defined as CD45⁻, Ter-119⁻, CD31⁻, CD105⁺ cells (Patra et al., 2018 (link)), 10⁶ cells from WT and Cko mice were stained as reported previously with fluorescent-tagged antibodies to CD45 (PE-Cy7) (BD Pharmingen, 552848), Ter-119 (PE-Cy7) (eBioscience, 25-5921), CD31 (BV421) (BD Pharmingen, 563356), and CD105 (Alexa-fluor 647) (BD Pharmingen, 562761), washed to remove unincorporated antibodies, and SSCs were FACS sorted at a density of one cell/well into a sterile 96-well plate containing lysis buffer. A total of two WT (SSC-WT1, SSC-WT2) and two Cko (SSC-Cko1, SSC-Cko2) mice were analyzed by RNA-Seq.