title: 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
design: 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
library_construction_protocol: 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.
identifiers.namespace: European Bioinformatics Institute
identifiers.id: E-MTAB-15734:Wt_oc_3_p