title: RN2
design: The transcriptome sequencing and subsequent analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China). Briefly, total RNAs from BMSCs and BMDMs were extracted utilizing the TRizol reagent (Invitrogen). The purity and concentration of the RNA were determined with a NanoDrop 2000 spectrophotometer (Thermo Scientific). RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies). Subsequently, libraries were prepared in accordance with the manufacturer's protocol using the VAHTS Universal V6 RNA-seq Library Prep Kit. The libraries were subjected to sequencing on an Illumina Novaseq 6000 platform, yielding 150 bp paired-end reads. Quality control of the mRNA-seq data was carried out using FastQC (v0.11.5) and the FASTX toolkit (0.0.13). Alignment to the Mus musculus reference genomes was performed using HISAT2 (v.2.0.4). Differentially expressed genes between shUsp7 and shNC groups were identified using the DEseq2 package (version 1.34.0) if they met the criteria of Q value < 0.05 and a fold change greater than 2 or less than 0.5. For Gene Set Enrichment Analysis (GSEA), gene sets related to innate immune response, Nod-like receptor protein 3 (NLRP3) inflammasome pathway, apoptotic cell clearance pathway, senescence-associated secretory phenotype (SASP) pathway, osteoblast differentiation, and apoptosis pathway were selected from an online GSEA database. The genes were ordered based on the degree of differential expression in the two types of samples. Subsequently, an assessment was conducted to determine whether the pre-defined gene set exhibited enrichment at either the top or bottom end of the ranking list. The statistical significance of whether a given gene set was enriched was assessed using GSEA analyses.