Fig. 1: The osteocyte transcriptome is broadly conserved across the skeleton.

a Schematic of the overall study design used to define the osteocyte transcriptome, to define an osteocyte transcriptome signature, the profile of genes enriched in osteocytes, and to identify their role in skeletal disease. b Diagram illustrating the 3 bone-types in which the osteocyte transcriptome was sequenced and compared. c Representative micro-CT and histological images of bone samples prior to processing (i–ii) and following osteocyte isolation (iii–iv), illustrating the effective removal of bone marrow, muscle and growth plates and enrichment of osteocytes (iv) (n = 8 biological independent mice). Boxes and dotted lines identify areas of high magnification confirming osteocyte isolation. Bone (B), bone marrow (BM), muscle (M) and growth plate (GP) are identified. d Actively expressed genes were identified based on the normalised gene expression distribution (blue histogram) and a hypothetical distribution of active promoters (red dotted line)²⁷ (i). Vertical line denotes the sample-specific threshold of actively expressed genes. Genes were considered active if they were above sample-specific threshold in all replicates, inactive if they were below the threshold in all replicates, or variable if they were above the threshold in at least one but not all replicates (ii). e The number of active genes and biotype composition in the osteocyte transcriptome across skeletal sites (i), and the percentage of total active genes detected in osteocytes in at least one (1+) and two or more (2+) orthogonal datasets (ii). The Any bone column reflects the number of genes actively expressed at any skeletal site. The All bones column reflects the number and proportion (as a percentage) of genes that were actively expressed in all skeletal sites. TEC = To be Experimentally Confirmed. f Pearson correlation of gene expression between individual replicates and bone types (mean of each bone type comparison represented by numbers above heatmap). Tib = tibia, Fem = femur, Hum = humerus, Rep = biological replicate. g Genes that were differentially expressed between osteocytes isolated from different bone types. Homeobox genes and antisense-RNAs are in red (FDR ≤ 0.05, LFC > 0.5). h Methodology for identifying genes expressed in a site-specific manner and a Venn diagram identifying the genes expressed in one bone type but not others.