RNA-Seq Analysis of FACS-sorted GFP-expressing and skeletal stem cells (SSC) from the bone marrow of mice with S1P ablation in the Osterix (Osx)-lineage (Cko)

Comparison of the transcriptome of GFP-expressing cells from the bone marrow compartment of Cko mice with the transcriptome of skeletal stem cells (SSC) from the bone marrow compartment of Cko and WT mice (homozygous floxed S1P mice). Overall design: Forelimbs and hindlimbs from mice were crushed in a mortar and pestle in the presence of phosphate buffered saline (PBS) and strained through a 70 micron filter and filtrate stored on ice. Crushed bones were digested with collagenase at 37 degree celcius for 15 min, strained through a 70 micron filter and pooled with the filtrate from above. The filtrate/cells were washed with cold PBS, and the RBCs lysed using an RBC lysis buffer (Sigma, R 7757) for 5 min. Following washings with cold PBS to remove lysed cells and hemoglobin, cells were suspended in PBS with 0.1% bovine serum albumin and stained with fluorescence-conjugated CD45, Ter-119, CD31, and CD105 antibodies and FACS-sorted for skeletal stem cells (SSC) defined as the population of cells positive for CD105 antigen but triple negative for Ter-119, CD31, and CD45 markers. Single cells were collected directly into 96 well plates from two WT (WT3 and WT5) and two Cko (Cko2 and Cko4) individually, lysed and RNAseq performed from RNA harvested from each well. Similarly, single GFP-expressing cells were directly FACS-sorted (i.e. without the use of any fluorescence conjugated antibodies) from the pooled bone marrow compartment of two Cko mice (in duplicate).