Project description:In this study, we aimed to systematically profile global RNA N6-methyladenosine (m6A) modification patterns in a mouse model of diabetic cardiomyopathy (DCM). Patterns of m6A in DCM and normal hearts were analyzed via m6A-specific methylated RNA immunoprecipitation followed by high-throughput sequencing (MeRIP-seq) and RNA sequencing (RNA-seq). A total of 973 m6A peaks were detected in DCM samples and 296 differentially methylated sites were selected for further study, including 106 hypermethylated and 190 hypomethylated m6A sites (fold change (FC) > 2, p < 0.05). Gene ontology and KEGG Pathway analyses indicated that unique m6A-modified transcripts in DCM were closely linked to cardiac fibrosis, myocardial hypertrophy, and myocardial energy metabolism. Overall, m6A modification patterns were altered in DCM, and modification of epitranscriptomic processes, such as m6A, is a potentially interesting therapeutic approach.
Project description:m7G-MeRIP-sequencing for 6 samples of the HPH (10% fractional inspired oxygen) and the control(N, 21% fractional inspired oxygen) groups.
Project description:We report the application of MeRIP sequencing technology for high-throughput profiling of RNA m6A modifications in wide-type and knock-down METTL3 or WTAP Human Umbilical Vein Endothelial Cells (HUVECs). Both the input samples without immunoprecipitation and the m6A IP samples were used for RNA-seq library generation with NEBNext® Ultra II Directional RNA Library Prep Kit (New England Biolabs, Inc., USA). Library sequencing was performed on an illumina Hiseq instrument with 150bp paired-end reads. Clean reads of all libraries were aligned to the reference genome (HG19) by Hisat2 software (v2.0.4). Methylated sites on RNAs (peaks) were identified by MACS software. Differentially methylated sites were identified by diffReps. And, guided by the Ensembl gtf gene annotation file, cuffdiff software (part of cufflinks) was used to get the gene level FPKM as the expression profiles of mRNA, and fold change and p-value were calculated based on FPKM, differentially expressed mRNA were identified. qRT-PCR validation was performed using SYBR Green assays. Finally, we find that METTL3/WTAP can regulate the expression level of target genes through m6A modification in HUVECs. This study provides a framework for applying MeRIP sequencing profiles to characterize vascular endothelial cells.
Project description:We report the first m6A RNA methylome of mouse islets using MeRIP sequencing. we recognized over 4000 methylation sites in 2-week islets.
