ACME dissociation: a versatile cell fixation-dissociation method for single-cell transcriptomics
Posted on: 6 July 2020
Preprint posted on 10 June 2020
Article now published in Genome Biology at http://dx.doi.org/10.1186/s13059-021-02302-5
Rediscovering a century-old technique to solve a modern single-cell biology problem: the case of ACME
Selected by Irepan Salvador-MartinezCategories: developmental biology, evolutionary biology
Introduction
In recent years, single-cell RNA seq (scRNAseq) studies are reshaping biology. The possibility to dissociate a tissue or even a whole animal and knowing the genes each cell was expressing has not only allowed the characterisation of known cell types, but also the discovery of new cell types.
Although scRNAseq technologies are under continuous improvement, a potential bottleneck for any scRNAseq study is the dissociation/fixation step. Current dissociation protocols use enzymatic or mechanical approaches that put live cells under stress for hours, with potential undesirable effects. A method that could fix and dissociate cells at once would therefore be highly beneficial for the single-cell community.
About the preprint
Garcia-Castro et al. tried to solve this problem by adapting a more than a century-old cell dissociation technique, originally called “maceration”. The maceration technique was first used on 1890, but continued to be used throughout the 20th century to dissociate cells from different soft-bodied animals such as cnidarians or planarians.
The adapted technique, named ACME by the authors after ACetic-MEthanol (referring to the solution used for the maceration, composed of acetic acid, methanol and glycerol), fixes single cells in suspension maintaining high integrity RNAs and dissociates them at the same time. Importantly, they showed that ACME-dissociated cells can be cryo-preserved at different check points during the process whilst retaining high integrity RNAs. This could open many opportunities, for example of doing cell dissociation of samples in the field and all downstream process in the lab.
Using the ACME protocol the authors managed to dissociate several animals, including zebrafish embryos, fruitfly larvae, spider embryos,annelid adults, snail larvae, and juvenile sea anemones. In these animals ACME cannot dissolve or penetrate hard parts like chorions, vitelline membranes, cuticles or shells, but applying a mechanical disruption and hard-part removal step was sufficient to extract their cells. In order to prove the usefulness of their technique for doing scRNAseq, the authors used it to sequence cells of 2 planarian species by combining ACME with SPLiT-seq (Rosenberg et al, 2018), a single-cell RNA-seq method that labels the cellular origin of RNA through combinatorial barcoding (Figure 1).
Results
The authors dissociated the cells of two planarian species, Schmidtea mediterranea and Dugesia japonica, and performed a species-mixing experiment (Figure 1) using SPLiT-seq. Planarians are soft-bodied flatworms (platyhelminthes) with remarkable regeneration capabilities: if you chop a planarian in three pieces, each piece will regenerate into a full worm in a couple of weeks.
In this single species-mixing experiment, the authors obtained ~14K and ~19K cells for D. japonica and for S. mediterranea, respectively. In order to analyse cell type composition, cells were clustered into cell types for each species. In the case of S. mediterranea (Figure 2) cell type composition was remarkably similar to a published cell atlas (Plass et al, 2018). For D. japonica, these results represent the first cell type atlas for this species. Using the homologues of S. mediterranea the authors could annotate the different cell types of D. japonica, finding that cell type proportions were similar in both species. Importantly, by producing a cell atlas of a second planarian (separated by ~85 my of evolution) the authors open the possibility of studying planarian cell type evolution.
Why I chose this preprint
I liked that the authors re-discovered an old technique for single-cell dissociation to solve a modern problem in single cell biology. I also liked the fact that as a proof of principle they created a new cell atlas for the planarian D. japonica, which will prove useful for studying cell type evolution in this group of animals.
Questions to the authors
Q1: Would ACME be suitable to fix/dissociate human cells from tissue biopsies? If so, I think it could be useful for collecting samples for biomedic studies (e.g. cancer).
Q2: Apart from SPLiT-Seq, which other cell barcoding techniques would be compatible with ACME?
References
Rosenberg AB, Roco CM, Muscat RA, et al. Single-cell profiling of the developing mouse brain and spinal cord with split-pool barcoding. Science 2018;360(6385):176-182.
Plass, M., et al., Cell type atlas and lineage tree of a whole complex animal by single-cell transcriptomics. Science, 2018. 360(6391).
doi: https://doi.org/10.1242/prelights.22652
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