Bone marrow macrophage meRIP sequencing

Total WT and FTO KO BMDM RNAs were extracted using a TRIzol reagent. RNA quality and quantity were determined with a Nanodrop TM OneCspectrophotometer and Qubit3.0 with the Qubit TM RNA Broad Range Assay kit. Fifty micrograms of total RNA were used for polyadenylated RNA enrichment using VAHTS mRNA capture beads. RNA fragments were incubated with an m6A-specific antibody for m6A immunoprecipitation. The stranded RNA sequencing library was constructed using the KC Digital TM Stranded mRNA Library Prep Kit for Illumina following the manufacturer instructions and sequenced on a NovaSeq 6000 sequencer with the PE150 model. Filtered MeRIP seq data were used for m6A site analysis. ExomePeak and deepTools software were used for peak calling, and peak distribution analysis, respectively. The differentiated m6A peaks were identified using a Python script. Sequence motifs enriched in m6A peak regions were verified using Homer. For RNA seq analysis, reads mapped to the exon regions of each gene were counted using featureCounts following RPKM value calculation. Analysis of differentially expressed genes and other analyses were performed using R 4.0.5. KEGG enrichment analysis was performed using KOBAS software.