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Polysome-profiling of IL4 or INFG polarized BMDMs

ABSTRACT: Polysome-profiling of IL4 or INFG polarized BMDMs

PROVIDER: PRJNA641348 | ENA |

REPOSITORIES: ENA

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Dataset's files

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			Action	DRS
	SRR12075278.fastq.gz	Fastqsanger.gz
	SRR12075279.fastq.gz	Fastqsanger.gz
	SRR12075280.fastq.gz	Fastqsanger.gz
	SRR12075281.fastq.gz	Fastqsanger.gz
	SRR12075282.fastq.gz	Fastqsanger.gz

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Polysome-profiling of IL4 or INFG polarized BMDMs

Project description:BMDMs from naïve female FVB mice were used to generate BMDMs which were polarized for 4 hours using either INFG or IL4. These were subjected to polysome-profiling.

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Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived IL4/IL13 treated BMDMs transcriptome profiling (RNA-seq) to quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods. Methods: BMDMs mRNA profiles of 8 weeks old wild-type (WT) mice were generated, stimulated with IL4/IL13, IL4/IL13+ TNF or TNF, deep sequenced, in triplicate, using Illumina NovaSeq 6000 platform. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9). Conclusions: Our study represents the first detailed analysis of IL4/IL13 or IL4/IL13+TNF stimulated BMDMs, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

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Project description:In order to assess whether differences in the transcriptomic profile of human tumor-associated macrophages vs. alveolar macrophages from adjacent non-tumor-tissue (GSE162669) were caused by factors secreted by tumor cells, primary human monocyte-derived macrophages (MDMs) were polarized towards a TAM-like phenotype by cultivating them in tumor cell-conditioned medium. A genome-wide comparison by bulk RNA-Seq confirmed a high similarity of ex vivo TAMs and in vitro polarized TAM-like macrophages. Other polarization schemes (M1: LPS/IFN-gamma, M2: IL10 or IL4) did not lead to similar transcriptomic changes. For details, see Hoppstädter et al., 2021 (doi:10.1016/j.ebiom.2021.103578).

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