Methanol fixation is the method of choice for droplet-based single-cell transcriptomics of neural cells

Abstract

Single-cell transcriptomics methods have become very popular to study the cellular composition of organs and tissues and characterize the expression profiles of the individual cells that compose them. The main critical step in single-cell transcriptomics is sample preparation. Several methods have been developed to preserve cells after sample dissociation to uncouple sample handling from library preparation. Yet, the suitability of these methods depends on the types of cells to be processed. In this project, we perform a systematic comparison of preservation methods for droplet-based single- cell RNA-seq (scRNA-seq) on neural and glial cells derived from induced pluripotent stem cells (iPSCs) and highlight their strengths and weaknesses. We compared the cellular composition and expression profile of single-cell suspensions from fresh NPCs with that of NPCs preserved with Dimethyl Sulfoxide (DMSO), Methanol, vivoPHIX and Acetil-methanol (ACME). Our results show that while DMSO provides the highest cell quality in terms of RNA molecules and genes detected per cell, it strongly affects the cellular composition and the expression profile of the resulting datasets. In contrast, methanol fixed samples display a cellular composition like that of fresh samples while providing a good cell quality and smaller expression biases. Taken together, our results show that methanol fixation is the method of choice for performing droplet-based single- cell transcriptomics experiments on neural cell populations.

FixNCut: single-cell genomics through reversible tissue fixation and dissociation

“…Meanwhile, the use of cold-active proteases on kidney samples resulted in fewer artifacts, but inefficient tissue dissociation [26]. In addition, studies have shown that neural cell populations (NCPs) suspension without methanol preservation also experiences alterations in cellular composition and gene expression [27]. For fragile tissue biopsies, such as the pancreas or skin, which contain delicate cell populations, cryopreservation and cellular dissociation steps may introduce biases on the cellular composition.…”

FixNCut: Single-cell genomics through reversible tissue fixation and dissociation

“…Meanwhile, the use of cold-active proteases on kidney samples resulted in fewer artifacts, but inefficient tissue dissociation (22). In addition, studies have shown that neural cell populations (NCPs) suspension without methanol preservation also experiences alterations in cellular composition and gene expression (23). For fragile tissue biopsies, such as pancreas or skin, which contain delicate cell populations, cryopreservation and cellular dissociation steps may introduce biases on the cellular composition.…”

Comparison of High-Throughput Single-Cell Rna-Seq Methods for Ex Vivo Drug Screening

“…The Parse and 10x Fixed methods offered the advantage of cell fixation, allowing for potential sample storage and collection at multiple time points. This feature may be particularly beneficial for studies involving delicate or easily disrupted cell types (41). On the other hand, the MULTI-seq protocol has the greatest potential for achieving ultra-high multiplexing (20) and thus lower costs, although it required a larger number of input cells and was more time-consuming compared to the other methods.…”