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Protocol for the purification and transcriptomic analysis of mouse astrocytes using GFAT

ABSTRACT: Astrocytes are glial cells of the central nervous system that modulate neuronal function. Here, we present glyoxal-fixed astrocyte nuclei transcriptomics (GFAT), a protocol for the purification and transcriptomic analysis of astrocyte nuclei from the cortex and cerebellum of adult and aged fresh mouse brain. We describe steps for tissue dissection, glyoxal fixation, homogenization, nuclei isolation, antibody staining, fluorescence-activated cell sorting, and RT-qPCR or bulk RNA sequencing. GFAT does not require transgenic lines or viral injection and allows parallel astrocyte and neuron profiling.

ORGANISM(S): Mus musculus

PROVIDER: GSE233509 | GEO | 2023/09/15

REPOSITORIES: GEO

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Project description:Within just a few years single cell RNA sequencing has become a frequently used standard method in many research fWithin just a few years single cell RNA sequencing has become a frequently used standard method in many research facilities worldwide. Declining sequencing costs and further refinements of the available protocols will pave the way for previously unimaginable prospects, up to single cell transcriptomic maps of entire organisms. The sample collection process can constitute a severe bottleneck in scRNA-seq experiments especially for solid tissues. Lengthy dissociation protocols are not uncommon to obtain sufficient amounts of starting material and can lead to significant changes in the gene expression profiles of cells. Preservation of the transcriptome prior to single cell dissociation can overcome this setback. Here we present an extensive performance analysis of glyoxal as an alternative fixative for scRNA-seq application. High numbers of transcripts and genes were recovered from glyoxal-fixed cells subjected to Drop-seq methodology with the best performance in Drosophila cells. While glyoxal fixation in Drosophila Kc167 and human HEK 293T cells revealed transcriptome data similar to the unfixed condition, reduced library complexity was observed for the human sample. We found the integrity of Drosophila intestinal tissue maintained following glyoxal fixation, while dissociation of the fixed tissue allowed sufficient cell isolation. In conclusion, we present glyoxal as a well-suited fixative for Drosophila samples that allows high-quality single cell transcriptomic analysis and successful intestinal tissue disaggregationcilities worldwide. Declining sequencing costs and further refinements of the available protocols will pave the way for previously unimaginable prospects, up to single cell transcriptomic maps of entire organisms. The sample collection process can constitute a severe bottleneck in scRNA-seq experiments especially for solid tissues. Lengthy dissociation protocols are not uncommon to obtain sufficient amounts of starting material and can lead to significant changes in the gene expression profiles of cells. Preservation of the transcriptome prior to single cell dissociation can overcome this setback. Here we present an extensive performance analysis of glyoxal as an alternative fixative for scRNA-seq application. High numbers of transcripts and genes were recovered from glyoxal-fixed cells subjected to Drop-seq methodology with the best performance in Drosophila cells. While glyoxal fixation in Drosophila and human cells revealed transcriptome data similar to the unfixed condition, reduced library complexity was observed for the human sample and might require further protocol optimization. We found the integrity of Drosophila intestinal tissue maintained following glyoxal fixation, while dissociation of the fixed tissue allowed sufficient cell isolation. In conclusion, we present glyoxal as a well-suited fixative for Drosophila samples that allows high-quality single cell transcriptomic analysis and successful intestinal tissue disaggregation.

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Protocol for the purification and transcriptomic analysis of mouse astrocytes using GFAT

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