ACME dissociation: a versatile cell fixation-dissociation method for single-cell transcriptomics

Abstract

Single-cell sequencing technologies are revolutionizing biology, but are limited by the need to dissociate fresh samples that can only be fixed at later stages. We present ACME (ACetic-MEthanol) dissociation, a cell dissociation approach that fixes cells as they are being dissociated. ACME-dissociated cells have high RNA integrity, can be cryopreserved multiple times, can be sorted by Fluorescence-Activated Cell Sorting (FACS) and are permeable, enabling combinatorial single-cell transcriptomic approaches. As a proof of principle, we have performed SPLiT-seq with ACME cells to obtain around ~34K single cell transcriptomes from two planarian species and identified all previously described cell types in similar proportions. ACME is based on affordable reagents, can be done in most laboratories and even in the field, and thus will accelerate our knowledge of cell types across the tree of life.In its original form, the maceration solution simply consisted of acetic acid and glycerol dissolved in water.Baguñà and Romero added methanol, as it speeds up dissociation [42]. Our protocol uses acetic acid and methanol, together with glycerol, dissolved in water. This solution produces fixed single cells in suspension with high integrity RNAs. Conveniently, we show that ACME-dissociated cells can be cryopreserved using DMSO [43] at different points throughout the process, with little detriment to their recovery and RNA integrity. As a proof of principle, we combined ACME to an optimised version of SPLiT-seq [34], including 4 rounds of barcoding, and were able to profile 33,827 cells from two different planarian species, Schmidtea mediterranea and Dugesia japonica, in a single run. We recover all S. mediterranea cell types from a previous study [13], at comparable proportions, showing that ACME dissociation does not introduce biases in cell type composition. Furthermore, we describe for the first time the single-cell transcriptome of D. japonica, opening the study of cell type evolution in this clade. Thus, in combination with SPLiT-seq or other approaches, ACME dissociation is a robust method to obtain high-quality single-cell transcriptomic data from fixed cells.

A resource of single‐cell gene expression profiles in a planarian Dugesia japonica

“…Although these genes play important roles in neoblast migration and differentiation, the cell types that express these genes remain elusive. While the previous SPLiT-seq (García-Castro et al, 2021) failed to detect DjMTA genes (Figure S2e), in this scRNA-seq data, DjMTA genes were expressed in the neoblasts (Djpi-wiA-positive cells) and neural cells (Djsyt-positive cells), and DjMTA-B…”

“…Although ( Sakurai et al, 2012), the cell type(s) expressing DjP2X-A remain unknown. The same as MTA genes, the previous SPLiT-seq (García-Castro et al, 2021) failed to detect DjP2X-A (Figure S2e).…”

“…Although there is only one publicly available large-scale scRNA-seq dataset of D. japonica (13,406 cells) (García-Castro et al, 2021), the numbers of detected transcripts are fewer than those of S. mediterranea; The average number of the detected genes and transcripts per cell are only 199 and 943, respectively (García-Castro et al, 2021), while those are 1,404 and 3,020 in a scRNA-seq dataset of S. mediterranea (Fincher et al, 2018).…”

A stony coral cell atlas illuminates the molecular and cellular basis of coral symbiosis, calcification, and immunity

“…In this context, our study shows the power and feasibility of single-cell analysis in species sampled from the wild. As exemplified here, and together with improvements in sampling strategies ( García-Castro et al, 2020 ), we anticipate that a phylogenetically rich animal cell type tree of life should be within reach in the coming years.…”

Single-cell atlases of two lophotrochozoan larvae highlight their complex evolutionary histories

“…S16). These genes have very low expression in adult planarian scRNA-seq (49) but are expressed at larval stages in the annelid Owenia fusiformis (50), suggesting that they may be specific to larval ciliary bands (fig. S16, C and D).…”