Cortical bone RNAseq comparison of WT, Sik2/3 DKO, and col1a1-Pth1r (H223R) mice. Cortical bone RNAseq comparison of WT, Sik2/3 DKO, and col1a1-Pth1r (H223R) mice

Cortical bone RNA was isolated from mice of the indicated genotypes followed by RNAseq Overall design: We analyzed n=6 WT, n=8 Sik2/3 DKO, n=11 WT, and n=12 col1a1-Pth1r H223R mutant samples. For long bone RNA isolation from control and SIK2/3 DKO animals, mice were sacrificed and both femurs were rapidly dissected on ice. Soft tissue was removed and epiphyses cut. Bone marrow cells were then removed by serial flushing of the marrow cavity with ice-cold PBS until bone appeared completely white. TRIzol (Life Technologies) was added to remaining bone tissue and samples were frozen at −80 °C and then homogenized. RNA was then extracted per the manufacturer's instructions, and further purified on RNeasy microcolumns (Qiagen). RNA with a A260/280 ratio 8.0 were used for downstream library construction. mRNAs were isolated by PAGE, followed by adaptor ligation and RT with PCR amplification. PCR products were again purified by PAGE and dissolved in EB solution. Double stranded PCR products were heat denatured and circularized by the splint oligo sequence. The ssCir DNA was formatted as the final sequencing library, and validated on bioanalyzer (Agilent 2100) prior to sequencing. The library was amplified with phi29 to general DNA nanoballs (DNBs) which were loaded into the patterned nanoarray followed by SE50 sequencing. On average we obtained 20M reads per bone RNA sample. Sequencing reads were mapped by the STAR aligner (86) to the mm9 reference genome using Ensembl annotation. Gene expression counts (CPM) were calculated using HTSeq v.0.6.0 (87). Differential expression analysis was performed using EgdeR package (88) based on the criteria of more than two-fold change in expression value versus control and false discovery rates (FDR) <0.05.