Rneasy midi spin columns

mRNA was isolated from adherent cells using 4 ml TRIzol and extracted using acid guanidinium thiocyanate-phenol-chloroform with GlycoBlue co-precipitant. mRNA was purified with Qiagen RNeasy Midi spin columns (Qiagen, Redwood City, CA) with on-column DNase digestion per the manufacturer’s protocol. cDNA was reverse transcribed from mRNA with Superscript III (Invitrogen, ThermoFisher) and quantitative RT-PCR reactions were conducted with 20 ng of template using custom primers (Table 1) and SYBR Select master mix (Applied Biosystems, Waltham, MA) for 50 cycles. Gene expression was calculated by normalizing to GAPDH (ΔCT) and then then to WT mice (ΔΔCT). Fold change was calculated as 2^−ΔΔCT.

For marrow ablation, intramedullary tissue was harvested by clipping the proximal and distal epiphyses and repeatedly reaming/flushing the diaphysis from both ends with 1 mL of TRIzol per animal using a 22 G needle and syringe. For mouse calvarial defects, a 4-mm biopsy punch was used to collect tissue centered over each defect, which was subsequently flash-frozen in liquid nitrogen in TRIzol and homogenized in a cryogenic bead mill (Bertin Precellys). For rat calvarial defects, a frontal quadrant of each day 5 sample was processed for gene expression analysis. RNA was isolated by acid guanidinium thiocyanate-phenol-chloroform extraction with GlycoBlue coprecipitant followed by purification with Qiagen RNeasy Midi spin columns with on-column DNase digestion per the manufacturer’s protocol, then reverse-transcribed. qPCR reactions were conducted with 20ng of template using custom primers (Table S1) and SYBR Select master mix for 40 cycles and analyzed using the 2^-ddCt method. Fold-change data is reported as mean ± standard deviation relative to basal tissue.