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NGS5523 - ATAC-seq WTAP and ZC3H13 KOs in hMDMS

ABSTRACT: NGS5523 - ATAC-seq WTAP and ZC3H13 KOs in hMDMS

PROVIDER: PRJNA1127605 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR29523900_1.fastq.gz	Fastqsanger.gz
	SRR29523900_2.fastq.gz	Fastqsanger.gz
	SRR29523901_1.fastq.gz	Fastqsanger.gz
	SRR29523901_2.fastq.gz	Fastqsanger.gz
	SRR29523902_1.fastq.gz	Fastqsanger.gz

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NGS5523 - ATAC-seq WTAP and ZC3H13 KOs in hMDMS

Project description:human primary macropahges (hMDMs) were genetically perturbed for WTAP and ZC3H13. ATAC-seq was performed 18h post IFNg (2ng/ml) or TNFa (10ng/ml) stimulation

2024-08-14 | GSE270634 | GEO

ZC3H13 and WTAP KO in hMDMs multi stimuli(NGS5421)

Project description:Here, we revealed an immunosuppressive role of m6A mediated by ZC3H13 and WTAP on TNF-driven inflammatory phenotype in human macrophages. In the following experiment we would like to investigate if the negative regulatory role of m6A is consistent across multiple stimuli using innate ligands (LPS, R848 and polyIC). We generated ZC3H13 KOs, WTAP KOs and NTC (WT) in monocyte-derived macrophages from 4 individual human donors and stimulated cells over night with LPS (5ng/ml) , R848 (50ng/ml) , polyIC (50ng/ml) and unstimulated cells as control. This yielded a total of 48 total samples.

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ZC3H13 and WTAP KO in hMDMs multi stimuli(NGS5421)

Project description:ZC3H13 and WTAP KO in hMDMs multi stimuli(NGS5421)

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NGS5413 - METTL3 and METTL14 KOs in hMDMs

Project description:NGS5413 - METTL3 and METTL14 KOs in hMDMs

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NGS5413 - METTL3 and METTL14 KOs in hMDMs

Project description:human primay macrophages (hMDMs) were genetically perturbed for METTL3 and stimulated with IFNg for 18h

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A novel RNA m6A modulator Zc3h13 plays an anchor role in facilitating nuclear RNA methylation and mouse embryonic stem cell self-renewal

Project description:N6-methyladenosine (m6A) is an abundant modification in eukaryotic mRNA, regulating mRNA dynamics by influencing mRNA stability, splicing, export and translation. Recent studies discovered m6A methyltransferases (?writer?), demethylases (?eraser?) and binding proteins (?reader?), which modulate m6A methylation. However, the precise m6A regulating machinery still remains incompletely understood. Here we demonstrate that ZC3H13, a zinc finger protein, plays an essential role in modulating m6A methylation on polyadenylated RNA in the nucleus. ZC3H13 exists in an evolutionary-conserved macromolecular complex containing WTAP, Virilizer and Hakai. We confirm the interaction among those proteins and demonstrate that knockdown of Zc3h13 in mouse embryonic stem cell (mESC) significantly decreases global m6A level on mRNA, mainly at 3? untranslated regions (3? UTR). Interestingly, fractionation assays show that upon Zc3h13 knockdown a great majority of WTAP, Virilizer and Hakai translocate to the cytoplasm and the nuclear presence of the methyltransferase Mettl3 and Mettl14 also decrease significantly. In contrast, knockdown of WTAP, Virilizer or Hakai does not change the nuclear localization of Zc3h13. This suggests that Zc3h13 is required for nuclear localization of the Zc3h13-WTAP-Virilizer-Hakai complex, which is important for RNA m6A methylation. Finally, Zc3h13 depletion, as does WTAP, Virilizer or Hakai, impairs self-renewal and triggers mESC differentiation. Taken together, our findings demonstrate that Zc3h13 plays an essential role in anchoring WTAP, Virilizer and Hakai in the nucleus to facilitate m6A methylation and to regulate mESC self-renewal.

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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.

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Binding motif analysis of METTL3，WTAP in METTL3，WTAP overexpressed Human 293T cells

Project description:label the cells overexpressed Myc tagged METTL3 and Flag tagged WTAP with 4-SU, the RNA bound by METT3,WTAP can be got by Myc or Flag IP followed by RNA isolation by using the TRIzol (Invitrogen) reagent by following the company manual.the RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol.The libraries were sequenced using HiSeq2000 (Illumina) in single-read mode, creating reads with a length of 50 bp. Sequencing chemistry v2 (Illumina) was used and samples were multiplexed in two samples per lane. discovery of the binding motif of METTL3，WTAP in METTL3，WTAP overexpressed Human 293T cells

2014-01-16 | E-GEOD-50580 | biostudies-arrayexpress

NGS5522 - hMDMS response to IFNg and TNFa

Project description:human primay macrophages (hMDMs) were unstimulated, stimulated with IFNg or costimulated with IFNg and TNFa for 18h

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mRNA profiles of WTAP siRNA-treated HUVECs

Project description:To examine the role of WTAP in splicing regulation, we performed high-throughput mRNA sequencing (RNA-seq) on RNA isolated from control, WTAP or Virilizer siRNA-treated HUVECs, yielding 12 million uniquely mapped 75nt pair-end tags from each sample. MapSplice software was used for differential expression and differences in transcript splice junctions . mRNA profiles of control, WTAP or Virilizer siRNA-treated HUVECs were generated by deep sequencing using Illumina GAII.

2014-09-25 | E-GEOD-41180 | biostudies-arrayexpress

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