A systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus RNA-Seq workflows

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.

Researchers from the University of Western Australia compare gene expression and cellular composition of single-cell suspensions prepared from adult mouse kidney using two tissue dissociation protocols. For each sample, they also compare fresh cells to cryopreserved and methanol-fixed cells. Lastly, the researcehers compare this single-cell data to that generated using three single-nucleus RNA sequencing workflows. Their 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, the researchers note an underrepresentation of T, B, and NK lymphocytes in the single-nucleus libraries.

Overview of experiments performed in this study

All experiments were carried out in biological triplicate using three kidneys from three different mice. a 37 °C dissociation used the Multi-tissue dissociation kit 2 from Miltenyi Biotec. b Cold dissociation was carried out on ice using B. Licheniformis protease. In a and b, methanol-fixed samples used 80% MeOH at − 20 °C and then were stored at − 80 °C. Cryopreservation was carried out using 50% FBS, 40% RPMI-1640, 10% and DMSO with gradient cooling to − 80 °C then stored in liquid nitrogen. c–e Whole kidneys were flash frozen using an isopentane bath − 30 °C and then stored at − 80 °C. Three different nuclei preparation methods were tested using either fluorescently activated nuclei sorting (FANS) or a sucrose gradient to enrich for singlet nuclei. f Bulk RNA-seq was carried out using the NEBNext Ultra II RNA Library Kit for Illumina with rRNA depletion or NEBNext Poly(A) mRNA isolation module.

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.