Systematic Assessment of Tissue Dissociation and Storage Biases in Single-Cell and Single-Nucleus RNA-Seq Workflows

Elena Denisenko1, Belinda B. Guo1, Matthew Jones1, Rui Hou1, Leanne de Kock1, Timo Lassmann2, Daniel Poppe1,3, Olivier Clément1, Rebecca K. Simmons1,3, Ryan Lister1,3, and Alistair R. R. Forrest1,$

  1. Correspondence to Alistair Forrest
  1. 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
  2. Telethon Kids Institute, Perth’s Children Hospital, the University of Western Australia, 15 Hospital Avenue, Nedlands, Perth, Western Australia, 6009, Australia
  3. 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

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.

Published Paper

Available from Genome Biology.

Sample Data

Available from the Gene Expression Omnibus (GEO) repository with the primary accession code GSE141115.