Published: 01-06-2022 11:09 | Updated: 02-06-2022 07:33

Improved method for single-cell RNA-sequencing at scale

Researchers at Karolinska Institutet have developed an improved single cell RNA sequencing (scRNA-seq) method to further enhance the ability to map cell types and genetic programs in health and disease. According to the researchers, the method, described in a paper in the journal Nature Biotechnology, offers a more affordable alternative to studying individual cells than current protocols without compromising data quality.

We have developed a scRNA-sequencing method called Smart-seq3xpress, which is a scalable and miniaturized implementation of Smart-seq3 that enables sensitive full-length scRNA-seq with molecular counting ability,” says Professor Rickard Sandberg, who co-authored the study with Michael Hagemann-Jensen and Christoph Ziegenhain from Department of Cell and Molecular Biology.  

Single-cell RNA-sequencing is routinely used to map cell types and genetic programs in health and disease. Most studies are currently carried out using droplet systems such as 10x Genomics and the dominant use of only one method has limited the potential for new discoveries. Smart-seq3xpress is on par in terms of cost with popular droplet-based sequencing methods that are limited to only sequencing the ends of RNA molecules.

Previously, full-length scRNA-seq has been substantially more expansive and time-consuming compared to for example droplet based scRNA-seq, and therefore infeasible to use in large-scale cell atlas projects.

The researchers from Rickard Sandberg's lab have now developed an improved single cell RNA sequencing (scRNA-seq) method that finally overcomes a long-standing compromise between cost, cellular throughput, and data quality.

10-fold reduction in cost

“Our miniaturized and streamlined protocol show a 10-fold reduction in cost and comparable price per cell to commercial droplet-based protocols,” says Rickard Sandberg. “Moreover, we show that Smart-seq3xpress profiling capture rare cell types that are often missed in 10x Genomics data, and we additionally obtain partial splicing information per cell type. With this method, we are finally able to overcome the long-standing compromise between cost, cellular throughput, and data quality in scRNA-sequencing.”

The potential to reduce costs by miniaturizing reaction volumes have been a long-standing goal although previous efforts by others to reduce volumes have come with declining assay quality. Successfully reducing the volumes became feasible when the researchers used hydrophobic overlay liquid that protects and encapsulates the tiny volumes during reaction.

"To showcase the data that can be generated with Smart-seq3xpress, we profiled 30,000 human PBMCs at unprecedented granularity and complexity, revealing cell-type specific splice variants," says Michael Hagemann-Jensen, research specialist in the Sandberg's lab.


Next step

Next the researchers want to utilize Smartseq3xpress to generate low-cost full-length transcriptome data at high cellular throughput in several tissues to map out the cell-type regulation in gene isoform expression.

“We are also using Smart-seq3xpress to investigate allelic expression patterns in clonal cells,” says Christoph Ziegenhain, postdoctoral researcher in the Sandberg's lab. “More broadly, the Smart-seq3xpress method have been inspiring for other methods in the lab that similarly have benefitted from reduced volumes for lower costs and higher throughputs, for example when measuring nascent RNAs in single cells."

A detailed step-by-step experimental protocol of Smart-seq3xpress is freely available for researchers through

This work was supported by grants from the Swedish Research Council, the Knut and Alice Wallenberg Foundation, the Göran Gustafsson Foundation and an EMBO long-term fellowship.


Scalable single-cell RNA sequencing from full transcripts with Smart-seq3xpress
Hagemann-Jensen M, Ziegenhain C, Sandberg R.
Nature Biotechnology May;30 10.1038/s41587-022-01311-4