Single-cell sequencing requires an individual cell per sample and therefore a large sample size is necessary to provide valid results. Reproducibility and sensitivity can be challenges for these single-cell assays in addition to the scalability and cost when large numbers of cells are analysed. Automation and miniaturisation can address these limitations. To assure high accuracy and precision, most library preparation protocols recommend volumes that are within the range of manual pipettes or that of large volume liquid handlers. However, only a small proportion of each of the libraries prepared will be required for the sequencing, therefore reducing the library prep volume will reduce costs.
This poster presents a low-cost and miniaturised method to create RNA sequencing libraries of single human pancreatic embryonic stem cells using mosquito® automated low-volume liquid handlers (TTP Labtech, Melbourn, UK). Technical and biological variability was compared between three different reaction volumes (2, 4 and 8 μL) and between four replicates.