Dr. Louise Laurent (University of California, San Diego, USA) and her team have published a miniaturized method for single-cell RNA library preparation that has reduced costs by 90% whilst increasing throughput and ensuring the high quality of libraries. (Sergio, MC et al. JALA 2016)!
In 1670, Antony van Leewenhoek made it possible to visualise a single cell under a microscope, now we have the potential to examine the entire genetic make-up of large populations of single cells simultaneously.
Advances in next-generation sequencing (NGS) methods have enabled whole transcriptome sequencing of single cells in high-throughput. A primary limitation of sequencing is cost. It is true that the cost of sequencing has now decreased, but this has not been the case for the library preparation step. If this part of the process could be miniaturized then the cost of library preparation could also be reduced. To achieve this, we have developed the ideal platform – reproducible, low-cost, low-volume benchtop liquid handlers designed for precious samples of varying viscosity!
the ‘how to’!
The published miniaturized method separates single cells into individual microchambers where the reverse transcription reaction generates high-quality cDNA. Using TTP Labtech’s mosquito LV genomics* liquid handler, as little as 20 pg of cDNA is used with the Nextera® XT library preparation kit (Illumina).
applying the new method!
To assess the quality of the libraries, Dr. Louise Laurent’s group applied this system to analyse pancreatic differentiation of human embryonic stem cells.
After differentiation, the cell cultures were dissociated to single-cell suspensions. cDNA libraries were generated from 20 pg of cDNA for three final reaction volumes (2-, 4- and 8- µL) in quadruplicate, in 384-well plates.
5-fold reduction of cDNA and 90% reduction in costs
The resulting single-cell RNA-seq data demonstrated that even at low reaction volumes it was possible to distinguish between cells at different stages of differentiation and also between individual cells within each stage (Fig 1). There was high reproducibility with a greater than 5-fold reduction of input cDNA in a 2 uL reaction volume. In addition, miniaturization provided total cost savings of 90%.
Fig 1. 2D principal component analysis demonstrates a clear separation between the libraries from each of the four cells. Importantly, the libraries did not cluster according to reaction volume, even within a single cell.
confirmed accurate and reproducible pipetting
mosquito provided accurate and reproducible pipetting for each of the steps in the sequencing process. In addition, TTP Labtech’s mosquito HV genomics** and 384 magnet (SZZ00136) enabled efficient bead clean-up of the libraries before they were sequenced on an Illumina HiSeq 2500.
“The workflow we have developed, in partnership with TTP Labtech, has the potential to provide researchers with significant cost savings while maintaining the quality of single cell sequencing. The instrument cost and their ease-of-use make the mosquito liquid handlers viable options for labs that would like to scale-down and automate next-generation sequencing sample preparation.” commented Dr. Laurent, Associate Professor, UCSD.
So if you want to save on sample and reduce costs but still want to analyse hundreds to thousands of single cells per project, then try using this method with TTP Labtech’s mosquito liquid handlers.
* & **: At the time of writing these instruments were known at mosquito HTS and mosquito HV.