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Straightforward extraction of high-quality RNA from glyoxal-fixed cells facilitates transcriptome analysis after cell staining and sorting by flow cytometry

ABSTRACT: We have developed a methodology for isolation of high-quality RNA from cells that are fixed, stained and sorted by flow cytometry that allows routine transcriptomic analysis of highly purified cell populations and single cells. This method, essentially developed by modifying existing staining and sorting protocols, involves fixation of cells with glyoxal, an alternative fixative to commonly used formaldehyde, followed by methanol permeabilization, a 2-step primary and secondary antibody staining and sorting by flow cytometry. The advantage of using glyoxal is that it does not crosslink RNA to proteins nor form stable RNA adducts, ensuring that RNA remains accessible and amenable to enzymatic manipulation after glyoxal fixation. The dataset comprising mRNA seq libraries from unprocessed or fixed and stained human cancer cells demonstrate that RNA recovered from glyoxal-fixed cells does not retain sufficient glyoxal adducts to impair reverse transcription, and also reveal very few differentially expressed genes between the 2 groups. The dataset derived from fixed and stained cells that were sorted into CCNB1 positive or negative fractions show the applicability of this method for sorting highly purified cell fractions as the CCNB1 positive fractions show strong enrichment for G2-phase cells according to the GO analysis.

ORGANISM(S): Homo sapiens

PROVIDER: GSE158177 | GEO | 2020/09/30

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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Straightforward extraction of high-quality RNA from glyoxal-fixed cells facilitates transcriptome analysis after cell staining and sorting by flow cytometry

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