Glyoxal as alternative fixative for single cell RNA sequencing

Abstract

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. Here, we developed and benchmarked protocols using glyoxal as a fixative for scRNA-seq application. Using Drop-seq methodology, we 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, we also found highly expressed genes overlapping to a large extent between experimental conditions. These results indicated that our fixation protocol did not induce considerable changes in gene expression and conserved the transcriptome for subsequent single cell isolation procedures. In conclusion, we present glyoxal as a suitable fixative for Drosophila cells and potentially cells of other species that allows high-quality scRNA-seq applications.

Practical Considerations for Single‐Cell Genomics

“…In split‐pool methods, fixation is critical to the barcoding step, and the fixation step may need to be optimized to improve library complexity and reduce background RNA contamination (Ma et al., 2020; Rosenberg et al., 2018). Paraformaldehyde solutions of 1%‐4% v/v are most commonly used, though alternative fixatives such as glyoxal (Bageritz et al., 2021) and dithio‐bis(succinimidyl propionate) (DSP, a.k.a. Lomant's reagent) (Attar et al., 2018) have been reported.…”