RNA m6A methylation in unilateral ureteral ligations model

Chronic kidney disease (CKD) is a high incidence disease. Renal fibrosis is the ultimate pathological pathway for chronic kidney diseases leading to end-stage renal failure. There is currently no effective treatment to prevent the renal fibrosis. Renal EMT is a key mechanism of renal fibrosis. Recent evidence suggests m6A modification in RNA can regulate EMT in tumor cells. However, a role of m6A RNA modification in renal fibrosis has not been reported. Therefore, we investigate whether m6A RNA methylation plays an important role in the epithelial-to-mesenchymal transition (EMT) process of renal fibrosis. Unilateral ureteral ligations were performed in C57BL/6 mice to establish a mouse model of renal fibrosis and TGF-b intervention was used in HK2 cells to establish a cellular model of renal fibrosis. The effects of m6A and the role of Mettl14 on kidney disease and HK2 cells were studied. 

Methods
Cloudseq Biotech Inc. (Shanghai, China) provided MeRIP-Seq service. Clean reads of all libraries were aligned to the reference genome by Hisat2 software (v2.0.4). Methylated sites on RNAs were identified by MACS software. Identified m6A peaks were subjected to motif enrichment analysis by HOMER, and metagene m6A distribution was characterized by R package MetaPlotR. Differentially methylated sites (fold change ≥2 and p < 0.05) were identified by diffReps. These peaks identified by both softwares overlapping with exons of mRNA were figured out and chosen by homemade scripts.