Early loss of bone mass in the SOD1(G93A) ALS mouse model is associated with loss of sensitivity to osteogenic factors and enhanced bone senescence
Early loss of bone mass in the SOD1(G93A) ALS mouse model is associated with loss of sensitivity to osteogenic factors and enhanced bone senescence
Bone tissue: After euthanasia, femurs were dissected and cleared of soft tissue. Articular cartilage was trimmed. Bone marrow was removed by PBS flushing and brief centrifugation. Bones were snap-frozen in liquid N₂ and kept on dry ice until processing. Primary osteoblasts: Long bones (femora/tibiae/humeri/radii-ulnae) were dissected, soft tissue removed, marrow flushed with PBS, and bone chips (1–2 mm²) prepared for outgrowth culture (see growth protocol). Primary osteoblasts were established as outgrowth cultures from long-bone chips. Chips were rinsed and plated in α-MEM supplemented with 15% FBS, 1% penicillin/streptomycin, and 1% L-glutamine. Cultures were maintained ~6 weeks with routine medium changes until ~80% confluence, then harvested for RNA. No experimental treatments were applied to RNA-seq samples. Cells were cultured under basal conditions as described in the growth protocol. Pulverized bone powder or cell pellets were lysed in guanidinium-based buffer (RLT + β-mercaptoethanol). Total RNA was extracted using Qiagen RNeasy with on-column DNase digestion, following the manufacturer's instructions. RNA quantity/quality were assessed by spectrophotometry and Bioanalyzer; only samples with RIN > 7 were used. Poly(A)-selected, strand-specific (second-strand/dUTP) mRNA libraries were prepared from 1 µg total RNA using an Illumina-compatible kit, according to the manufacturer's protocol. Adapter-ligated cDNA was PCR-amplified with limited cycles, size-selected for ~300–500 bp inserts, and QC'd by Bioanalyzer and qPCR.