GSE133988
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 <1.7 was not used for downstream analysis, this workflow allows us to avoid use of samples with excessive bone matrix protein contamination. For cDNA synthesis, 1?µg RNA was used in synthesis reactions according to the instructions of the manufacturer (Primescript RT, Takara). SYBR Green-based quantitative PCR (qPCR) detection was performed using FastStart Universal SYBR Green (Roche, Basel, Switzerland) on a StepOne Plus (Applied Biosystems, Carlsbad, CA) thermocycler. All PCR primer sequences are listed in Supplemental Table 3. Transcript levels for genes of interest were determined relative to housekeeping gene (ß-actin) using standard methods (84). A similar protocol for cortical bone isolation was used for the comparison of effects of sclerostin antibody treatment versus vehicle. For bone RNA isolation from control and C1HR mice, the same protocol was used with the exception that the marrow flushing step was not performed. Due to overt phenotypic difference between control and experimental (SIK2/3 DKO, C1HR transgenics, Scl-Ab treated) mice, we acknowledge that many differentially expressed genes here may be due to changes in cellular tissue composition. Therefore, we focus on similarities and differences across comparisons (WT/SIK2/3 DKO versus WT/C1HR, WT/SIK2/3 DKO versus control/Scl-Ab, WT/C1HR versus control/Scl-Ab), to determine the overall molecular concordance between the three different models of increased trabecular bone mass.  RNA-seq   RNA sequencing from bone RNA samples for Figure 4 was conducted using a BGISEQ500 platform (BGI, Shenzhen, China) (85). Briefly, RNA samples with RIN values >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.