Mettl3-mediated m6A RNA methylation regulates the fate of bone marrow mesenchymal stem cells and osteoporosis

The aim is to analyze the transcriptome profiling (RNA-seq) of Mettl3 knockout mesenchymal stem cells (MSCs) and identify m6A modified mRNAs (m6A-seq). For RNA-seq, total RNAs from MSCs of Prx1-Cre;Mettl3fl/fl mice and their control littermates were extracted and purified using poly-T oligo-attached magnetic beads. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations, and were then subjected to Illumina HiSeq 2500. We used FastQC (v0.11.5) and FASTX toolkit (0.0.13) to control the quality of RNA-seq data and mapped them to Mus musculus reference genomes using HISAT2 (v.2.0.4). Ballgown software (v.3.4.0) was performed to identify differentially expressed genes and transcripts. Genes were considered significantly differentially expressed if showing ≥ 2.0 fold change and P value < 0.05. For m6A-seq, total RNAs were isolated from MSCs and then mRNAs were purified using poly-T oligo-attached magnetic beads. The purified mRNAs were fragmentated and used for immunoprecipitation with anti-m6A antibody, then the pulled down fragments were purified and used for libraries construction with the Illumina TruSeq Stranded mRNA Sample Prep Kit following the manufacturer's instructions. m6A libraries were sequenced with Illumina NextSeq500. Sequencing reads were aligned to mm9 using tophat and peak calling was performed with MACS2. The mRNA profiles of WT and Prx1-Cre;Mettl3fl/fl MSCs were generated by deep sequencing, in duplicate, using Illumina HiSeq 2500. m6A profiles of WT MSCs were sequenced with Illumina NextSeq500.