RNA-Seq analysis of Kdm6bf/f and Col2a1-CreERT2;Kdm6bf/f primary chondrocytes

Purpose: The aims of this study were to identify differentially expressed genes between Kdm6bf/f and Col2a1-CreERT2;Kdm6bf/f primary chondrocytes. Methods: RNA samples of primary chondrocytes were prepared from Kdm6bf/f and Col2a1-CreERT2;Kdm6bf/f mice and were sequenced and analyzed by Shanghai Novel Bioinformatics Co, Ltd. Before read mapping, clean reads were obtained from the raw reads by removing the adaptor sequences, reads with >5% ambiguous bases (noted as N) and low-quality reads containing more than 20 percent of bases with qualities of <13. The clean reads were then aligned to the mouse genome (version: hg19_GRCh37) using the MapSplice program (v2.1.6). We applied EBseq algorithm to filter the differentially expressed genes (DEGs), after the significant analysis and FDR analysis under the following criteria: i) Fold Change>2 or <0.5; ii) FDR<0.05. DEGs were used for gene ontology (GO) and pathway analysis. We downloaded the GO annotations from NCBI, UniProt and the Gene Ontology. Fisher's exact test was applied to identify the significant GO categories and FDR was used to correct the p-values. Pathway analysis was used to determine the significant pathway of the differentially expressed genes according to the KEGG database. The Fisher's exact test was utilized to select the significant pathway, and the threshold of significance was defined by P-value and FDR. Conclusions: Our study represents the first detailed analysis of the role of Kdm6b in joint cartilage development and homeostasis, which provided a new therapeutic modalities for OA treatment. Overall design: Kdm6bf/f and Col2a1-CreERT2;Kdm6bf/f primary chondrocytes were treated with 4-hydroxytamoxifen (4OH-TM) (1 µM) for 48 hours, and then RNA samples were prepared from these cells for RNA-Seq.