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MeRIP sequencing reveals angiogenic properties of vascular endothelial cells

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE142386 | GEO | 2019/12/20

REPOSITORIES: GEO

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