Mixer mill

For quantitative real-time PCR analysis (RT-qPCR) of WBV cortical bone, collected tibiae were cleaned from connective tissue, their shafts were cut-out and flushed with sterile PBS to restrict the sampling to cortical bone, then crushed in liquid nitrogen with steel balls in a mixer mill (Sartorius, Gottingen, Germany) before extraction with Tri-Reagent (Sigma-Aldrich). For HG experiment, the tibia shafts were flushed with Tri-Reagent in order to best sample the trabecular compartment. RNA harvested with Tri-Reagent was purified on columns (Rneasy Plus Mini Kit, Qiagen, Hilden, Germany), quantified with the Ribogreen kit (Invitrogen, Life Technologies, Eugene, OR, USA) and quality checked with an Experion automated electrophoresis station (Bio Rad, Hercules, CA, USA). Messenger RNA was reverse transcribed (iScript cDNA synthesis Kit, Biorad) and 400 ng of cDNA were amplified using the SYBR Green I dye (Lightcycler faststart DNA masterSYBR green I, Roche, Germany). Names and primer sequences of the genes of interest are given in Table S1. Expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) did not vary significantly within or between groups (not shown).