Tutorial – High-Throughput Single-Cell RNA-Seq of Cells and Nuclei

Harnessing RNA-Seq to Create Gene Expression Profiles

– from Genetic Engineering News – Jennifer Chew, Adam Bemis, Ronald Lebofsky, Ph.D., Anna Quinlan, Ph.D. , Kelly Kaihara, Ph.D.

Complex biological systems rely on the coordinated activities of individual cells. The function of these cells can differ drastically, even within a single tissue, but capturing this heterogeneity is difficult with conventional technologies that treat millions of cells as a single sample.

These approaches yield averages that mask important cell-to-cell transcriptional differences and hide the activities of subpopulations that can be crucial to tissue health and development. To better understand the contribution of individual cells in human health and to develop more targeted treatments for disease, it is important to characterize human biology at the single-cell level.

mRNA-FISH, single-cell RT-qPCR, and single-cell RNA sequencing (RNA-seq) have emerged as popular single-cell transcriptomics technologies. We briefly discuss the pros and cons of each of these methods and demonstrate that the Illumina® Bio-Rad® Single-Cell Sequencing Solution overcomes many of the challenges of currently available platforms by allowing high-throughput and scalable sequencing of even the most challenging sample types, including large cells and small species such as nuclei.


Single cells are easily isolated and sequenced using the scalable Illumina Bio-Rad Single-Cell Sequencing Solution. UMI: unique molecular identifier.

Limitations of Traditional Single-Cell Technologies

mRNA-FISH is a microscopy-based method that uses fluorescently labeled probes to count individual transcripts of interest in fixed cells or tissues. Unlike other single-cell approaches, mRNA-FISH provides spatial information, but like other single-cell methods, mRNA-FISH is limited in the number of genes that can be analyzed in a single experiment. Because each target mRNA species needs to be assigned a spectrally distinct fluorophore, the number of genes that can be interrogated simultaneously is small.

Single-cell RT-qPCR allows investigation of a larger number of genes. When a 96-well plate is used, this method allows analysis of 1–10 genes in up to 96 cells. Throughput can be increased to 96 genes across 96 cells by using microfluidic chip-based experiments. This method, however, provides insight into just a tiny fraction of the transcriptionally active genes in a given cell.

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