Single cell RNA sequencing (scRNA-seq) has become an important method to identify cell types, delineate the trajectories of cell differentiation in whole organisms and understand the heterogeneity in cellular responses. Nevertheless, sample collection and processing remain a severe bottleneck for scRNA-seq experiments. Cell isolation protocols often lead to significant changes in the transcriptomes of cells, requiring novel methods to preserve cell states.
Researchers from the German Cancer Research Center have developed and benchmarked protocols using glyoxal as a fixative for scRNA-seq application. Using Drop-seq methodology, the researchers detected high numbers of transcripts and genes from glyoxal-fixed Drosophila cells after scRNA-seq. The effective glyoxal fixation of transcriptomes in Drosophila and human cells was further supported by a high correlation of gene expression data between glyoxal-fixed and unfixed samples. Accordingly, they also found highly expressed genes overlapping to a large extent between experimental conditions. These results indicated that this fixation protocol did not induce considerable changes in gene expression and conserved the transcriptome for subsequent single cell isolation procedures.
Effect of glyoxal fixation on cell integrity, RNA quality and purity
(a) Workflow to assess quality of glyoxal-fixed human and Drosophila cell lines for single cell RNA transcriptome studies. Single cell suspension from different species were mixed equally, subjected to Drop-Seq and their transcriptome compared by downstream analysis. (b) Proportion of reads mapped to coding, UTR (untranslated region), intronic, and intergenic regions. Two biological replicates were analyzed per condition for each cell line. (c) Fraction of mitochondrial transcripts out of total mRNA transcripts per condition and cell line. (d) Percentage of mixed cell barcodes detected per data set. Horizontal line indicates the mean.