Rneasy minelute spin column from the

For isolation of the tibial articular cartilage, knee joints were disarticulated allowing articular cartilage to be shaved from the tibial plateau and immediately frozen over dry ice. For isolation of femur heads, hip joints were disarticulated and femur heads cut from the femoral neck with a bone scissor prior to freezing over dry ice. Using a Bullet Blender Gold (Next Advance), the tissues were homogenized in TRIzol (Thermo Fisher) with a mix of 2, 1, and 0.5 mm RNase-free zirconium oxide beads (Next Advance) for 10 minutes. Phases were separated using chloroform per TRIzol instructions, and the aqueous layer and one volume of 70% ethanol transferred to a RNeasy MinElute Spin Column from the RNeasy Micro Kit (QIAGEN). RNA was isolated per Micro Kit instructions.

Using scalpels, articular cartilage was shaved from the tibial plateau and frozen over dry ice. Using the Bullet Blender Gold (Next Advance), the cartilage was homogenized in 250 μl of TRIzol (Thermo Fisher) with a mix of 2, 1, and 0.5 mm RNase-free zirconium oxide beads (Next Advance) for 10 minutes. Phases were separated using 100 μl chloroform per TRIzol manufacturer’s instructions (Thermo Fisher), and the aqueous layer and one volume of 70% ethanol were transferred to a RNeasy MinElute Spin Column from the RNeasy Micro Kit (QIAGEN). mRNA was then isolated per Micro Kit instructions (QIAGEN).