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M6A MeRIP-Seq of liver in Mettl3flox/flox and hepatocyte-specific Mettl3 knockout (HKO) mice

ABSTRACT: Purpose: Through mRNA m6A-profiling, we aim to characterize the mRNA m6A changes in the liver obtained from Mettl3flox/flox and HKO mice. Methods: Total RNA was extracted using Tripure Isolation Reagent (Roche, Mannheim, Germany) from liver of Mettl3 flox/flox and HKO mice at 8 weeks old. Each sample (300 μg total RNA) was pooled from 3 mice for each group. Poly(A)+ RNA was purified using Dynabeads™ mRNA Purification Kit (Invitrogen) following the manufacturer’s instructions. Chemically fragmented poly(A)+ RNA was incubated with m6A antibody (Synaptic System, 202003) for immunoprecipitation following the standard protocol of Magna MeRIPTM m6A Kit (MERCK, 17-10499). Enrichment of m6A mRNA was then analyzed by high-throughput sequencing using Illumina Hiseq X platform. The m6A peaks were detected by MACS2. The motif search was detected by MEME and DREME. Conclusion: The mRNA m6A profiles in the liver of Mettl3flox/flox and HKO mice were characterized.

ORGANISM(S): Mus musculus

PROVIDER: GSE142835 | GEO | 2021/10/29

REPOSITORIES: GEO

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m6A MeRIP-Seq of interscapular brown adipose tissue (iBAT) in Mettl3flox/flox and BAT-specific Mettl3 knockout (BKO) mice

Project description:Purpose: Through mRNA m6A-profiling, we aim to characterize the mRNA m6A changes in iBAT obtained from Mettl3flox/flox and BKO mice. Methods: Total RNA was extracted using Tripure Isolation Reagent (Roche, Mannheim, Germany) from iBAT of Mettl3 flox/flox and BKO mice at 8 weeks old. Each sample (300 μg total RNA) was pooled from 5 mice for each group. Poly(A)+ RNA was purified using Dynabeads™ mRNA Purification Kit (Invitrogen) following the manufacturer’s instructions. Chemically fragmented poly(A)+ RNA was incubated with m6A antibody (Synaptic System, 202003) for immunoprecipitation following the standard protocol of Magna MeRIPTM m6A Kit (MERK, 17-10499). Enrichment of m6A mRNA was then analyzed by high-throughput sequencing using Illumina Hiseq X platform. The m6A peaks were detected by MACS2. The motif search was detected by HOMER. Conclusion: The iBAT mRNA m6A profiles in Mettl3flox/flox and BKO mice were characterized.

2020-02-06 | GSE141076 | GEO

m6ARIP-Seq of liver in Wtapflox/flox and hepatocyte-specific Wtap knockout (HKO) mice

Project description:Purpose: Through mRNA m6A-profiling, we aim to characterize the mRNA m6A changes in the liver obtained from Wtapflox/flox and Wtap-HKO mice. Methods: Total RNA was extracted using Tripure Isolation Reagent (Roche, Mannheim, Germany) from liver of Wtap flox/flox and Wtap-HKO mice at 8 weeks old. Each sample (300 μg total RNA) was pooled from 5 mice for each group. Two independent biological replicates for each group were used for m6ARIP-seq. Poly(A)+ RNA was purified using Dynabeads™ mRNA Purification Kit (Invitrogen) following the manufacturer’s instructions. Fragmented mRNA was incubated with m6A antibody (Synaptic System, 202003) for immunoprecipitation. Then, immunoprecipitated mRNAs or Input was used for library construction with NEBNext ultra RNA library prepare kit for Illumina (New England Biolabs).The library preparations were sequenced on an Illumina Novaseq 6000 platform with a paired-end read length of 150 bp according to the standard protocols. The m6A peaks were detected by exomePeak R package (version 2.16.0). The motif search was detected by HOMER(version 4.9.1). Conclusion: The mRNA m6A profiles in the liver of Wtapflox/flox and Wtap-HKO mice were characterized.

2022-06-02 | GSE192884 | GEO

m6A MeRIP-Seq of interscapular brown adipose tissue (iBAT) in Wtapflox/flox and BAT-specific Wtap knockout (BKO) mice

Project description:Purpose: Through mRNA m6A-profiling, we aim to characterize the mRNA m6A changes in iBAT obtained from Wtapflox/flox and Wtap-BKO mice. Methods: Total RNA was extracted using Tripure Isolation Reagent (Roche, Mannheim, Germany) from iBAT of Wtap flox/flox and Wtap-BKO mice at 8 weeks old. Each sample (300 μg total RNA) was pooled from 8 mice for each group. Poly(A)+ RNA was purified using Dynabeads™ mRNA Purification Kit (Invitrogen) following the manufacturer’s instructions. Fragmented mRNA was incubated with m6A antibody (Synaptic System, 202003) for immunoprecipitation. Then, immunoprecipitated mRNAs or Input was used for library construction with NEBNext ultra RNA library prepare kit for Illumina (New England Biolabs).The library preparations were sequenced on an Illumina Novaseq 6000 platform with a paired-end read length of 150 bp according to the standard protocols. The m6A peaks were detected by exomePeak R package (version 2.16.0). The motif search was detected by HOMER(version 4.9.1). Conclusion: The iBAT mRNA m6A profiles in Wtapflox/flox and BKO mice were characterized.

2022-10-19 | GSE202668 | GEO

MeRIP-Seq of pancreatic islets in Mettl3flox/flox and islet β cell-specific Mettl3 knockout (β-Mettl3-KO) mice

Project description:Purpose: Through mRNA m6A-profiling, we aim to characterize the mRNA m6A changes in pancreatic islets obtained from Mettl3flox/flox and β-Mettl3-KO mice. Methods: Total RNA was extracted using Tripure Isolation Reagent (Roche, Mannheim, Germany) from pancreatic islets of Mettl3flox/flox and β-Mettl3-KO mice at 8 weeks old. A total of 2-3 μg RNAs were pooled from nine Mettl3flox/flox mice and twelve β-Mettl3-KO mice, respectively. Three independent biological replicates of each group were used for MeRIP-seq. Fragmented RNA (~100 nt) was incubated for 2 hr at 4oC with anti-m6A polyclonal antibody (Merk Millipore) in the immunoprecipitation experiment. Then, immunoprecipitated RNAs or Input was used for library construction with Ovation SoLo RNA-Seq System Core Kit (NuGEN). The library preparations were sequenced on an Illumina Novaseq platform with a paired-end read length of 150 bp according to the standard protocols. After mapping reads to the reference genome, exomePeak R package (version 2.16.0) was used for the m6A peak identification in each anti-m6A immunoprecipitation group with the corresponding input samples serving as a control, and q-value threshold of enrichment of 0.05 was used for all data sets. The m6A-enriched motifs of each group were identified by HOMER (version 4.9.1). Conclusion: The islet mRNA m6A profiles in Mettl3flox/flox and β-Mettl3-KO mice were characterized.

2020-11-13 | GSE155614 | GEO

m6A MeRIP-Seq of liver in Mettl3flox/flox and hepatocyte-specific Mettl3 knockout (HKO) mice

Project description:m6A MeRIP-Seq of liver in Mettl3flox/flox and hepatocyte-specific Mettl3 knockout (HKO) mice

| PRJNA598574 | ENA

RNA-seq analysis of mRNA profiles in the livers of Mettl3 flox/flox and hepatocyte-specific Mettl3 knockout (HKO) mice

Project description:Purpose: The goal of this study is to compare transcriptome profilings of liver from Mettl3 flox/flox and hepatocyte-specific Mettl3 knockout (HKO) mice. Methods: Total RNA was extracted using Tripure Isolation Reagent (Roche, Mannheim, Germany) from livers of Mettl3 flox/flox and HKO mice at 8 weeks old. mRNA profiles were generated by deep sequencing using an Illumina HiSeq X Ten platform. Paired-end clean reads were aligned to the mouse reference genome(Ensemble_GRCm38.90) with Hisat2 (version 2.0.4), and the aligned reads were used to quantify mRNA expression by using HTSeq (version 0.9.1). Conclusion: The hepatic mRNA profiles in Mettl3 flox/flox and HKO mice were characterized.

2021-10-29 | GSE141216 | GEO

RNA-seq analysis of mRNA profiles in the livers of Wtapflox/flox and hepatocyte-specific Wtap knockout (HKO) mice

Project description:Purpose: The goal of this study is to compare transcriptome profilings of liver from Wtapflox/flox and hepatocyte-specific Wtap knockout (HKO) mice. Methods: Total RNA was extracted using Tripure Isolation Reagent (Roche, Mannheim, Germany) from livers of Wtap flox/flox and Wtap-HKO mice at 8 weeks old. mRNA profiles were generated by deep sequencing using an Illumina Novaseq platform. Paired-end clean reads were aligned to the mouse reference genome(Ensemble_GRCm38.p6) with TopHat (version 2.0.12), and the aligned reads were used to quantify mRNA expression by using HTSeq-count (version 0.6.1). Conclusion: Our study represents the first detailed analysis of hepatic mRNA profiles in Wtapflox/flox and Wtap-HKO mice at 8 weeks old, generated by RNA-seq technology. Our results showed that 3486 genes were downregulated, and 3706 genes were upregulated. Gene ontology (GO) analysis showed that downregulated genes were associated with small molecule catabolic process, fatty acid metabolic process, amino acid metabolic process, steroid metabolic process, cholesterol metabolic process, xenobiotic metabolic process, fatty acid beta-oxidation, alcohol metabolic process, and glucose metabolic process, whereas the upregulated genes were associated with wound healing, leukocyte migration, chemotaxis, and positive regulation of cytokine production.

2022-06-02 | GSE168850 | GEO

ATAC-seq of liver samples in Mettl3 flox/flox and hepatocyte-specific Mettl3 knockout (HKO) mice

Project description:Purpose: The goal of this study is to determine whether hepatic deletion of Mettl3 affects chromatin accessibility. Methods: Liver samples were pooled from livers of Mettl3 HKO and Mettl3 flox/flox mice (n=4 for each group). Nuclei was extracted from liver samples, and the nuclei pellet was resuspended in the Tn5 transposase reaction mix. The transposition reaction was incubated at 37°C for 30 min. Equimolar Adapter1 and Adatper 2 were added after transposition, PCR was then performed to amplify the library. After the PCR reaction, libraries were purified with the AMPure beads and library quality was assessed with Qubit. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufactuer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina NovaSeq 6000 platform and 150 bp paired-end reads were generated. ATAC-seq analysis was performed using a standard protocol. Results: The hepatic chromatin accessibility in Mettl3 flox/flox and HKO mice were characterized.

2021-10-29 | GSE141245 | GEO

Title: RNA-seq analysis of mRNA profiles in the livers of HuR flox/flox and hepatocyte-specific HuR knockout (HKO) mice fed with an MCD for 3 weeks.

Project description:ionPurpose: The goal of this study is to investigate how HuR regulates NASH progression. Methods: The hepatic mRNA profiles of HuR flox/flox and hepatocyte-specific HuR knockout (HKO) mice (n=3 for each group) fed with an MCD for 3 weeks were generated by deep sequencing using an Illumina Novaseq platform. Paired-end clean reads were aligned to the mouse reference genome(Ensemble_GRCm38.p6) with TopHat (version 2.0.12), and the aligned reads were used to quantify mRNA expression by using HTSeq-count (version 0.6.1). Conclusion: Our study represents the first detailed analysis of hepatic mRNA profiles in HuR flox/flox and hepatocyte-specific HuR knockout (HKO) mice fed with an MCD for 3 weeks, generated by RNA-seq technology. Our results show that 353 genes were upregulated and 422 genes were downregulated. Gene Ontology (GO) analysis showed that genes related to leukocyte migration, leukocyte chemotaxis, cell chemotaxis, neutrophil migration and Fc receptor signaling pathway were significantly decreased, which contributes to the protective effects of NASH progression in HKO mice.

2023-06-17 | GSE152670 | GEO

Project description:RNA-Seq of Flox and IRF6-HKO mice liver

| PRJNA522920 | ENA

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