Systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus RNA-seq workflows
Authors: Alistair R R Forrest
Daniel Poppe
Matthew Jones
Leanne de Kock
Belinda B Guo
Olivier Clément
Rebecca K Simmons
Timo Lassmann
Elena Denisenko
Rui Hou
Ryan Lister
Journal: Genome biology (2020)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: 10x Genomics; RNA-seq; Single-cell transcriptomics; scRNA-seq; snRNA-seq.
Affiliations: Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, the University of Western Australia, PO Box 7214, 6 Verdun Street, Nedlands, Perth, Western Australia, 6009, Australia., Telethon Kids Institute, Perth's Children Hospital, the University of Western Australia, 15 Hospital Avenue, Nedlands, Perth, Western Australia, 6009, Australia., Australian Research Council Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, the University of Western Australia, 35 Stirling Hwy, Crawley, Perth, Western Australia, 6009, Australia., Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, the University of Western Australia, PO Box 7214, 6 Verdun Street, Nedlands, Perth, Western Australia, 6009, Australia. .

Abstract:  Background: Single-cell RNA sequencing has been widely adopted to estimate the cellular composition of heterogeneous tissues and obtain transcriptional profiles of individual cells. Multiple approaches for optimal sample dissociation and storage of single cells have been proposed as have single-nuclei profiling methods. What has been lacking is a systematic comparison of their relative biases and benefits. Results: Here, we compare gene expression and cellular composition of single-cell suspensions prepared from adult mouse kidney using two tissue dissociation protocols. For each sample, we also compare fresh cells to cryopreserved and methanol-fixed cells. Lastly, we compare this single-cell data to that generated using three single-nucleus RNA sequencing workflows. Our data confirms prior reports that digestion on ice avoids the stress response observed with 37 °C dissociation. It also reveals cell types more abundant either in the cold or warm dissociations that may represent populations that require gentler or harsher conditions to be released intact. For cell storage, cryopreservation of dissociated cells results in a major loss of epithelial cell types; in contrast, methanol fixation maintains the cellular composition but suffers from ambient RNA leakage. Finally, cell type composition differences are observed between single-cell and single-nucleus RNA sequencing libraries. In particular, we note an underrepresentation of T, B, and NK lymphocytes in the single-nucleus libraries. Conclusions: Systematic comparison of recovered cell types and their transcriptional profiles across the workflows has highlighted protocol-specific biases and thus enables researchers starting single-cell experiments to make an informed choice. 

Retracted: No

PubPeer comments:
 This paper is on a really important practical matter, and it is great that such a systematic experiment was designed for this purpose. However, to talk about *Systematic assessment of tissue dissociation and storage biases*, one needs to assess the **quality of the cells (Read depth, UMI and gene counts)**, library complexity, and fraction of reads in cells u2013 not only proportions of cell types. Basically any such analysis seem to be missing from the paper, which thus only shows one side of the coin. It would be great if the authors could comment on this matter, and provide some additional insight, helping people to decide for a method of choice. ,