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RNA methylation in diabetes [MeRIP-seq]

ABSTRACT: N6-methyladenosine (m6A) is one of the most prevalent and abundant epigenetic modifications in various fundamental bioprocesses. We hypothesized that m6A-mediated inflammation pathway contributes to diabetes. Total RNA was extracted from the retinas of wide type rat and their littermates with diabetes by STZ injection. A total amount of 1 μg RNA per sample was used as an input for the RNA sample preparations. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia), the reagents used in library preparation are NEBNext® Ultra RNA Library Prep Kit for Illumina (NEB, USA) .After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated. The MeRIP-seq was carried out in Novogene (Beijing, China). Briefly, 2 μg total RNA was extracted from the retinas of both wide type mice and their littermates with diabetes. The integrity and concentration of extracted RNA was detected using an Agilent 2100 bioanalyzer (Agilent) and simpliNano spectrophotometer (GE Healthcare), respectively. Fragmented RNA (~100 nt) was incubated for 2 hours at 4 ℃ with anti-m6A polyclonal antibody (Synaptic Systems) in the immunoprecipitation experiment. Then, immunoprecipitated RNA or input was used for library construction with Ovation SoLo RNA-Seq System Core Kit (NuGEN). The library preparations were sequenced on Illumina Novaseq platform with a paired-end read length of 150 bp according to the standard protocols. The sequencing was carried out with three independent biological replicates.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE176353 | GEO | 2021/07/01

REPOSITORIES: GEO

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Project description:Purpose: To understand the biological function of citratemt accumulation, we performed RNA-seq analysis. Methods:MLE12 cells were transfected simultaneously with shIdh3α lentivirus (MOI: 50) and shSlc25a1 lentivirus (MOI: 50) and negative control (shCtrl lentivirus, MOI: 50) in the presence of 1HitransG P (Lot: REVG005, GeneChem).Empty construct was used as control for all experiments. The cells were washed with fresh complete medium 16 h later and then cultivated for an additional 80 h.Total RNA was extracted from control and Slc25a1 plus Idh3α-deficient MLE12 cells. The transcriptome library for sequencing was generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® following the manufacturer’s recommendations. The index codes were added to attribute sequences to each sample. After the quality of sequencing libraries was confirmed on the Agilent 2100 system, clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia, San Diego, CA, USA) according to the manufacturer’s instructions. The library preparations were then sequenced on an Illumina Hiseq 2500 platform (Illumina, San Diego, CA, USA), and paired-end reads were generated. Transcriptome assembly was accomplished based on the left.fq and right.fq using Trinity with min_kmer_cov set to 2 by default and all other parameters set to default. Results: Using an optimized data analysis workflow,We found that 215 genes were upregulated in MLE12 cells with citratemt accumulation . To determine the functional properties of the top 30 genes upregulated by citratemt, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. KEGG pathway analysis indicated that the 30 genes were involved in regulating the TNF signaling pathway , one of the critical signaling pathways for triggering necroptosis Conclusions: These results further suggest that citratemt accumulation specifically drives necroptosis

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RNA methylation in diabetes [MeRIP-seq]

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