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Pooled CRISPR Screening Identifies m6A as a Positive Regulator of Macrophage Activation [m6A-RIP-seq]

ABSTRACT: Total RNAs were isolated from WT_CON, KO_CON, WT_LPS and KO_LPS Raw 264.7 cells, subjected to standard m6A RIP protocol. The libraries were generated with IPed and Input RNAs and subjected to Sequencing. Raw sequencing reads were aligned to the mouse genome (mm10) with Tophat , and gene expression levels were measured by Cufflinks.

ORGANISM(S): Mus musculus

PROVIDER: GSE162254 | GEO | 2021/04/30

REPOSITORIES: GEO

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Next-generation sequencing measuring m6A enrichment in CD4+ T cells isolated from WT mice

Project description:CD4+ T cells were isolated by StemCell CD4+ T cell isolation kit of spleen and lymph nodes WT mice. Total RNAs were isolated from the pure CD4+ T cells, subjected to standard m6A RIP protocol. The libraries were generated with IPed and Input RNAs and subjected to sequencing. Raw sequencing reads were aligned to the mouse genome (mm10) with Tophat, and gene expression levels were measured by Cufflinks.

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Pooled CRISPR Screening Identifies m6A as a Positive Regulator of Macrophage Activation [RNA-seq]

Project description:The involvement of m6A modification in macrophage activation has been validated in our study that the expression of TNF-α in Mettl3-depleted Raw 264.7 cells stimulated with LPS were markedly reduced in comparison to control cells. To further explore the biological effects of m6A deficiency macrophages, we performed RNA sequencing analysis of Mettl3-KO and WT control Raw 264.7 cells upon LPS treatment. The GO enrichment analysis documented that the downregulated transcripts in Mettl3-KO Raw 264.7 cells were enriched in innate immune response related to defense and external stimulus. Notably, transcripts of the downstream components of the TLR4 signaling pathway, such as proinflammatory cytokines (Tnf-α, Il-6, Il-1β, Il-18,and Il-23) and co-stimulation molecules (Cd86), were downregulated in Mettl3-deficient cells, suggesting that METTL3 has a critical function in controlling the innate immune response of Raw 264.7 macrophages.

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