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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.

Project description:We show that high quality microarray gene expression profiles can be obtained following FACS sorting of cells using combinations of transcription factors. We use this transcription factor FACS (tfFACS) methodology to perform a genomic analysis of hESC-derived endodermal lineages marked by combinations of SOX17, GATA4, and CXCR4, and find that triple positive cells have a much stronger definitive endoderm signature than other combinations of these markers. Additionally, SOX17+GATA4+ cells can be obtained at a much earlier stage of differentiation, prior to expression of CXCR4+ cells, providing an important new tool to isolate this earlier definitive endoderm subtype. Overall, tfFACS represents an advancement in FACS technology which broadly crosses multiple disciplines, most notably in regenerative medicine to redefine cellular populations. 21 Total samples were analyzed. Samples collected after 5 days of hESC differentiation included SOX17+GATA4+ CXCR4+ cells, unfixed CXCR4+ cells, and unsorted fixed cells. Samples collected after 3 days of differentiation included SOX17+GATA4+ cells, SOX17-GATA- cells, and unsorted fixed cells. As controls, we analyzed both unfixed hESCs and fixed hESCs. All samples contained biological duplicates, triplicates or quadriplicates. We performed hierarchical clustering to demonstrate whether cellular fixation alone could change gene expression. We based this analysis on 1647 transcript clusters with coefficient of variation > 0.5 across the samples and expression values >= 500 in at least 2 out of the 21 samples. We found that fixation and staining steps did not cause distortions in gene expression measurements. This is supported by the fact that fixed and unfixed cells cluster together based upon differentiation stage, not based upon degree of fixation. Furthermore, using GSEA analysis, we found that the SOX17+GATA4+CXCR4+ day 5 cells and day 3 SOX17+GATA4+ were more enriched for compiled gold-standard endodermal genes than the CXCR4+ day 5 population.

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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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