We found that Rb inactivation induces sphere-forming activity under 3D culture condition in primary soft tissue sarcoma cells derived from p53 null mice. To elucidate molecular mechanism, we employed RNA-sequence as a discovery platform to identify gene set of Rb-depeltion induced sphere-forming cells. To enrich sphere-forming cells and make the culture condition equivalent to that of parental cells (Primary cells), we relplated sphere-forming cells on normal dishes (Rb shRNA Secondary cells), and obtained total RNA soon after replating. This gene set was highly enriched in genes involved in inflammation, steroid biosynthesis and central carbon metabolisms. Non-target Secondary cells were derived from surviving control cells without sphere-formation under 3D culture condition, and used as control for manipulation of culture condition.