RNA-seq of peripheral whole blood of patient E pre-pamidronate treatment
Children aged 16 years or younger with a diagnosis of CNO who met the Bristol Criteria and were due to start intravenous pamidronate treatment at the Bristol Royal Hospital for Children between August 2022 and November 2023 were included.  Peripheral blood samples (2.5mL) were collected in PAXgenes bottles (PreAnalytiX/Qiagen) immediately before and 3-5 months after first pamidronate cycle. Samples were stored at room temperature for up to 6h and then frozen at -20C (<48 hours) before transfer to -70C.  Total RNA was extracted and purified from blood samples using the PAXgene Blood RNA Kit (PreAnalytiX/Qiagen) as per manufacturers instructions. RNA quality was assessed by RNA TapeStation (Agilent) and Nanodrop (Thermo Scientific). Libraries were prepared with the Illumina Stranded mRNA library preparation kit (Illumina). Paired-end sequences from NextSeq500 sequencer (Illumina) were quality trimmed and adaptor sequencers were removed with Fastp V0.12.4. Reads were mapped to the human reference genome build GRCh38 with STAR V2.7.9a and counts per gene were obtained by HTSeq-count V0.11.3. Normalisation, principal component analysis, and differential expression was calculated with with DESeq2 V1.40.2. Participant identity was included in statistical modelling to account for repeated sampling of the same individual. Adjusted p-values were corrected for multiple testing by False Discovery Rate (FDR) method. Gene set enrichment analysis not restricted to differentially expressed genes was performed using Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) terms in clusterProfiler V4.8.3 with org.Hs.eg.db V3.17.0.