Single-cell RNA sequencing to study vascular diversity and function

Abstract

Purpose of review: Single-cell RNA sequencing (scRNA-seq) can capture the transcriptional profile of thousands of individual cells concurrently from complex tissues and with remarkable resolution. Either with the goal of seeking information about distinct cell subtypes or responses to a stimulus, the approach has provided robust information and promoted impressive advances in cardiovascular research. The goal of this review is to highlight strategies and approaches to leverage this technology and bypass potential caveats related to evaluation of the vascular cells.

Recent findings: As the most recent technological development, details associated with experimental strategies, analysis, and interpretation of scRNA-seq data are still being discussed and scrutinized by investigators across the vascular field. Compilation of this information is valuable for those using the technology but particularly important to those about to start utilizing scRNA-seq to seek transcriptome information of vascular cells.

Summary: As our field progresses to catalog transcriptomes from distinct vascular beds, it is undeniable that scRNA-seq technology is here to stay. Sharing approaches to improve the quality of cell dissociation procedures, analysis, and a consensus of best practices is critical as information from this powerful experimental platform continues to emerge.

Figure 1.. Cell dissociation and single-cell preparation of vascular cells.

A,B) Two isolation methods used to capture and enrich for the cells in the tunica intima. A) The aortae are dissected and enzymatically digested, followed by cell sorting based on subjective markers. The sorted cells are then used to generate libraries. B) The aortae are dissected and cut through the dorsal portion of the segmented wall opening the tubular structure as a flat plan (en face). The aortae are then pinned to a silicone dish with the tunica intima facing up and washed with versene and incubated with trypsin for 5 minutes. After trypsin, the tunica intima is gently scraped using a feather scalpel and the cells are collected and used for library generation. C) Isolation method used to enrich vascular smooth muscle cells (vSMCs). Mice are injected with heparin, perfused with versene, and then aortae are dissected and digested using a liberase enzyme cocktail. Single-cell suspension is then used to generate libraries. tSNE plots are representative of the cell types captured using each method.

Figure 2.. Quality control measurements executed during scRNA-seq.

A) Prior to library generation, quality control (QC) measurements of cell viability and enrichment (i.e. using hemocytometers, flow cytometry, microscopy etc.) are critical for downstream single-cell quality. B) Post-library construction QC using a High sensitivity TapeStation or a Bioanalyzer, as well as measuring library concentration with Qubit, will shed light on the quality of the library prior to paying for sequencing. C) Quality assessment of scRNA-seq data is essential before downstream analysis. Assessing the sequencing saturation, the number of cells, genes, and mitochondrial genes detected, are a few QC post-sequencing metrics used.