Experimental Considerations for Single-Cell RNA Sequencing Approaches

Abstract

Single-cell transcriptomic technologies have emerged as powerful tools to explore cellular heterogeneity at the resolution of individual cells. Previous scientific knowledge in cell biology is largely limited to data generated by bulk profiling methods, which only provide averaged read-outs that generally mask cellular heterogeneity. This averaged approach is particularly problematic when the biological effect of interest is limited to only a subpopulation of cells such as stem/progenitor cells within a given tissue, or immune cell subsets infiltrating a tumor. Great advances in single-cell RNA sequencing (scRNAseq) enabled scientists to overcome this limitation and allow for in depth interrogation of previously unexplored rare cell types. Due to the high sensitivity of scRNAseq, adequate attention must be put into experimental setup and execution. Careful handling and processing of cells for scRNAseq is critical to preserve the native expression profile that will ensure meaningful analysis and conclusions. Here, we delineate the individual steps of a typical single-cell analysis workflow from tissue procurement, cell preparation, to platform selection and data analysis, and we discuss critical challenges in each of these steps, which will serve as a helpful guide to navigate the complex field of single-cell sequencing.

Diverse homeostatic and immunomodulatory roles of immune cells in the developing mouse lung revealed at single cell resolution

“…To comprehensively define the lung immune landscape at birth, we isolated whole lungs from C57BL/6 (B6) mice at four stages of perinatal development: the early saccular (E18.5), late saccular (P1) early alveolar (P7) and late alveolar stages (P21) ( Figure 1A), and quantified gene expression by scRNA-Seq. Lung tissue was isolated, the pulmonary vasculature perfused to remove circulating immune cells, and the tissue digested using an in-house optimized protocol to ensure maximal cell viability as published protocols 18,19 induced high amount of cell death in the embryonic and early postnatal lung (Supplemental Figure 1). Live CD45+ cells were sorted by FACS, processed by Smart-seq2 and sequenced on Illumina NovaSeq 6000 ( Figure 1A).…”

Personalized medicine—concepts, technologies, and applications in inflammatory skin diseases

“…Optimally, a time course experiment (multiple samples taken at different time points) should be performed to investigate the expression profiles' temporal dependence (60,61), which is also of importance for the selection of biomarkers, as some may display transient expression, while others are more stable, and therefore more robust in a clinical setting. Just as for DNA sequencing, single-cell RNA-sequencing (scRNA-seq) is now opening a window to the cellular phenotype, as it allows for unprecedented detail analysis of cellular heterogeneity and development (62,63). Finally, novel in situ sequencing techniques such as fluorescent in situ sequencing of RNA (FISSEQ) (64) and STARmap (65) allow for determination of the actual, 3-dimensional location of gene expression in cells and tissues (66).…”

Single-cell analysis of intestinal immune cells during helminth infection

“…Recent advances in single cell analysis have significantly increased our understanding of multiple diseases and cell types in different tissues 1,2 . However, many of these technologies require single cell suspensions as an input, which limits our assessment of difficult-to-process tissues [2][3][4] . One prominent example is the intestine, which is at the center of many research questions that focus on nutrient uptake 5 , host-microbiome interactions [6][7][8] , local and systemic immune tolerance [9][10][11] and gastrointestinal diseases and infections [12][13][14][15][16] , but represents a challenging tissue to digest 17,18 .…”