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Meso-Seq for in-depth transcriptomics in ultra-low amounts of FACS-purified neuronal nuclei

ABSTRACT: Meso-Seq for in-depth transcriptomics in ultra-low amounts of FACS-purified neuronal nuclei

PROVIDER: PRJNA768272 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR16173800.fastq.gz	Fastqsanger.gz
	SRR16173801.fastq.gz	Fastqsanger.gz
	SRR16173802.fastq.gz	Fastqsanger.gz
	SRR16173803.fastq.gz	Fastqsanger.gz
	SRR16173804.fastq.gz	Fastqsanger.gz

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Meso-Seq for in-depth transcriptomics in ultra-low amounts of FACS-purified neuronal nuclei

2022-08-11 | GSE185221 | GEO

Meso-seq for in-depth transcriptomics in ultra-low amounts of FACS-purified neuronal nuclei.

Project description:Profiling of gene expression in sparse populations of genetically defined neurons is essential for dissecting the molecular mechanisms that control the development and plasticity of neural circuits. However, current transcriptomic approaches are ill suited for detailed mechanistic studies in sparse neuronal populations, as they either are technically complex and relatively expensive (e.g., single-cell RNA sequencing [RNA-seq]) or require large amounts of input material (e.g., traditional bulk RNA-seq). Thus, we established Meso-seq, a meso-scale protocol for identifying more than 10,000 robustly expressed genes in as little as 50 FACS-sorted neuronal nuclei. We demonstrate that Meso-seq works well for multiple neuroscience applications, including transcriptomics in antibody-labeled cortical neurons in mice and non-human primates, analyses of experience-regulated gene programs, and RNA-seq from visual cortex neurons labeled ultra-sparsely with viruses. Given its simplicity, robustness, and relatively low costs, Meso-seq is well suited for molecular-mechanistic studies in ultra-sparse neuronal populations in the brain.

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Comparison of microglial proteomes of MACS and FACS-based purified CD11b+ microglia

Project description:Background Proteomic characterization of microglia has been limited by low yield and contamination by non-microglial proteins by magnetic-activated cell sorting (MACS) enrichment strategies. To determine whether a fluorescent-activated cell sorting (FACS)-based strategy overcomes these limitations, we compared microglial proteomes of MACS and FACS-based purified CD11b+ microglia in order to identify core sets of microglial proteins in adult mouse brain tissue. Results Quantitative multiplex proteomics by tandem mass tag mass spectrometry (TMT-MS) of MACS-enriched (N = 5) and FACS-purified (N = 5) adult wild-type CD11b+ microglia identified 1,791 proteins, of which 953 were differentially expressed indicating significant differences between both approaches. While the FACS-purified microglia proteome was enriched with cytosolic, endoplasmic reticulum and ribosomal proteins involved in protein metabolism and immune system functions, the MACS-enriched microglia proteome was enriched with proteins related to mitochondrial function and synaptic transmission. As compared to MACS, the FACS microglial proteome showed strong enrichment for canonical microglial proteins while neuron, astrocyte, and oligodendrocyte proteins were decreased. Interestingly, we observed enrichment of endothelial specific proteins in the FACS microglia proteome. By comparing FACS-purified microglia proteomes with transcriptomes, we observed highly concordant as well as highly discordant proteins that were abundant at the protein level but low at the transcript level. Conclusions We demonstrate that TMT-MS proteomics of FACS-purified adult microglia is superior to column-based enrichment approaches, resulting in purer and highly-enriched microglial proteomes. We also define core sets of highly-abundant adult microglial proteins including Moesin (Msn) that can guide future studies.

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