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Identification of Rbm25-binding RNAs in bone-marrow-derived macrophages by RIP-seq

ABSTRACT: Identification of Rbm25-binding RNAs in bone-marrow-derived macrophages by RIP-seq

PROVIDER: PRJNA1002386 | ENA |

REPOSITORIES: ENA

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			Action	DRS
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	SRR25526924_1.fastq.gz	Fastqsanger.gz
	SRR25526924_2.fastq.gz	Fastqsanger.gz

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Identification of Rbm25-binding RNAs in bone-marrow-derived macrophages by RIP-seq

Project description:To identify the potential mRNAs that bound by Rbm25, which might be the direct target of Rbm25 for alternative splicing in murine macrophages.

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H3K9ac and H3K27ac Cut&Tag profiling of wild type and Rbm25-deficient Bone Marrow Derived macrophage

Project description:Purpose: To transcriptome widely reveal histone acetylation modification H3K9ac and H3K27ac regulation by Rbm25, we performed Cleavage Under Targets and Tagmentation (CUT&Tag) analysis of wild-type (WT) and Rbm25-deficiency BMDMs. Methods: Mouse BMDMs were generated from bone marrow cells in RPMI-1640 medium with recombinant mouse M-CSF (20ng/ml). BMDMs were stained to confirm the surface expression of CD11b and F4/80. Cells with purity >97.5% were used for subsequent experiments. WT and Rbm25 deficient BMDMs were stimulated with LPS (100ng/ml) for 4 hours. Results: We carried out CUT&Tag assays using antibody against H3K9ac or H3K27ac, and found the global influence of Rbm25 in regulating H3K9ac and H3K27ac of transcription regulatory regions of key genes for inflammatory macrophage effector function.

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Next Generation Sequencing Facilitates Quantitative Analysis of wild type and Rbm25-deficient Bone Marrow Derived macrophage Transcriptomes

Project description:Purpose: The purpose of this study is to detect activated or silenced genes during wild type (WT) and Rbm25-deficient bone marrow derived macrophages (BMDMs). Gene expression differences between two samples could be found using transcriptome profiling (RNA-seq) analysis. Methods: Mouse BMDMs were generated from bone marrow cells in RPMI-1640 medium with recombinant mouse M-CSF (20ng/ml). BMDMs were stained to confirm the surface expression of CD11b and F4/80. Cells with purity >97.5% were used for subsequent experiments. WT and Rbm25 deficient BMDMs were stimulated with LPS (100ng/ml) for 0, 2 or 8 hours, of which RNA profiles were generated by deep sequencing, using Illumina. Results: We mapped about 10 million sequence reads per sample to the mouse genome, identified hundreds of genes with significant mRNA variation between WT and Rbm25 deficient BMDMs.

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Next Generation Sequencing Facilitates Quantitative Analysis of wild type and Rbm25-deficient Bone Marrow Derived macrophages

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Project description:Data from the immunoprecipitation of CEBPB from bone marrow derived macrophages.

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Project description:To reveal PTBP1-bound RNAs, we conducted RNA immunoprecipitation sequencing (RIP-seq) with total RNA samples from MKN-28 cells.

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Efficient, specific and combinatorial control of endogenous exon splicing with dCasRx-RBM25

Project description:Efficient targeted control of exon splicing is a major goal of functional genomic and therapeutic applications. Guide RNA-directed, deactivated (d)Cas CRISPR enzymes fused to splicing effectors represent a promising strategy due to the flexibility and presumed specificity of these systems. However, efficient, selective, and generalizable activation of targeted endogenous exons using this approach has not been reported. Here, we identify dCasRx-RBM25 as a potent activator of exons by screening over 300 dCasRx splicing factor fusions tethered to splicing reporters. dCasRx-RBM25 also strongly activates splicing of endogenous alternative exons, when recruited to downstream intron sequences using single guide RNAs. In transcriptome wide analyses we observe a high degree of specificity of dCasRx-RBM25 for endogenous exon targeting. We further leverage the guide array-processing activity of dCasRx to simultaneously target multiple endogenous exons for activation and repression by dCasRx-RBM25. Our results pave the way for versatile exon-resolution functional assays and splicing-directed therapeutic applications.

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Project description:RIP-seq analysis of PTBP1-bound RNAs

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Conditioned medium from bone marrow derived macrophages

Project description:Conditioned medium (CM) from bone marrow derived macrophages untreated or treated with LPS was collected and filtered through a 0.22-μm filter. The filtered CM was sequentially fractionated with 50-kDa and 100-kDa Amicon filters. The 50–100 kDa fraction of CM was analyzed by mass spectrometry.

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