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RNA methylation in diabetes [RNA-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: GSE176352 | GEO | 2021/07/01

REPOSITORIES: GEO

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RNA methylation in diabetes [MeRIP-seq]

Project description: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.

2021-07-01 | GSE176353 | GEO

RNA-seq of human ovarian adenocarcinoma cell line SKOV3 treated with bisphenol A (10 or 100 nM) against untreated controls

Project description:Human ovarian adenocarcinoma SKOV3 cells were exposed to BPA (10 or 100 nM) or 0.1% DMSO for 24 h,and then total RNA was extracted from cells using Trizol reagent. Sequencing libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Then, the index-coded samples were clustered on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hisreq 4000 platform with 150 bp paired-end reads.

2018-05-04 | E-MTAB-6295 | biostudies-arrayexpress

Assay for transposase-accessible chromatin-sequencing (ATAC-seq) of liver samples in Wtapflox/flox and hepatocyte-specific Wtap knockout (Wtap-HKO) mice

Project description:Purpose: The goal of this study is to determine whether hepatic deletion of Wtapaffects chromatin accessibility. Methods: Each liver sample was pooled from three Wtap-HKO and Wtapflox/flox mice, respectively. Three independent biological replicates of each group were used for ATAC-seq. 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 Wtapflox/flox and Wtap-HKO mice were characterized.

2022-06-02 | GSE168945 | 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

Transcriptome Sequencing Facilitates Quantitative Analysis of BMDM (bone marrow induced macrophages) from FBXW7f/f and LysM-cre FBXW7f/f Transcriptomes before or after LPS treatment

Project description:Purpose: The goals of this study are to compare genes change between FBXW7f/f and LysM-cre FBXW7f/f BMDM after LPS treatment Methods: BMDM mRNA from 6-8weeks FBXW7f/f (WT) and LysM-cre FBXW7f/f (KO) mice were generated by transcription sequencing, using Illumina. 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 manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and 125 bp/150 bp paired-end reads were generated.

2020-02-20 | GSE125242 | 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

Analysis of changes in transcriptomes of the rat medial prefrontal cortex tissues (mPFC) in vehicle group, chronic mild stress (CMS) group, and CMS+Phloretin group

Project description:Purpose: The goals of this study are to investigate the effects of Phloretin (PHL) on the changes of genes in rat mPFC tissues between Vehicle group, CMS group, and CMS+PHL group. Methods: Vehicle group: Rats received vehicle by oral gavage for 2 weeks, and then were left undisturbed for 3 weeks except for necessary procedures, including handling and routine cleaning for 3 weeks. CMS group: Rats received vehicle by oral gavage for 2 weeks, and then were subjected to eight different stressors for 3 weeks in a random order, including foot shock, water deprivation, cold water swimming, food deprivation, restraint, clip tail, light/dark cycle reversal, and cage tilt. CMS+PHL group: Rats were pre-treated with 20 mg/kg. Phloretin for 2 weeks, and then exposed to above eight stressors randomly together with 20 mg/kg Phloretin for 3 weeks. In each group, we collected mPFC tissues from 3 rats and total RNA was extracted. RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system. 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 manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated. Results: We found that Phloretin treatment changed some gene expression, especially in neuronal plasticity, compared with CMS group.

2023-02-22 | GSE193284 | 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

Expression data from Head and neck squamous cell carcinoma cell lines cell line Cal27 after TCF19 knockout mediated by CRISPR/CAS9

Project description:The deletion of TCF19 in CAL27,oral squamous cell carcinoma cell line,was conducted by using CRISPR/Cas9 system. For WT and TCF19 KO CAL27 cell lines, samples were done in triplicates. A total of 3μg of high-quality RNA per sample was used for ribosomal RNA removal by the Epicentre Ribo-zero rRNA Removal Kit (Epicentre, USA) and the sequencing library was prepared using the rRNA-depleted RNA by the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer’s recommendations. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina, San Diego, CA, USA), followed by the 150-bp paired-end sequencing on the HiSeq X Ten instrument (Illumina, San Diego, CA, USA) according to the manufacturer’s protocols.

2021-01-01 | GSE153354 | GEO

Transcriptome-wide N6-methyladenosine modification profiling in patients with recurrent implantation failure (RIF)

Project description:Purpose:using m6A modified RNA immunoprecipitation sequence (m6A-seq), to establish the m6A methylation transcription profiles in recurrent implantation failure (RIF). Methods: GenSeq® Low Input Whole RNA Library Prep Kit (GenSeq, Inc.) was used to construct RNA libraries for IP and input samples by following the manufacturer's instructions. The library quality was evaluated using Agilent 2100 bioanalyzer and then sequenced in a NovaSeq platform (Illumina 6000). Result:using m6A modified RNA immunoprecipitation sequence (m6A-seq), methylated sites in mRNA,lncRNA and circRNA are identified. Conclusions: Our study revealed the m6A methylation landscape by MERIP sequencing.

2022-10-16 | GSE205398 | GEO

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