Miniaturization Technologies for Efficient Single-Cell Library Preparation for Next-Generation Sequencing.

As the cost of next-generation sequencing has decreased, library preparation costs have become a more significant proportion of the total cost, especially for high-throughput applications such as single-cell RNA profiling. Here, we have applied novel technologies to scale down reaction volumes for library preparation. Our system consisted of in vitro differentiated human embryonic stem cells representing two stages of pancreatic differentiation, for which we prepared multiple biological and technical replicates. We used the Fluidigm (San Francisco, CA) C1 single-cell Autoprep System for single-cell complementary DNA (cDNA) generation and an enzyme-based tagmentation system (Nextera XT; Illumina, San Diego, CA) with a nanoliter liquid handler (mosquito HTS; TTP Labtech, Royston, UK) for library preparation, reducing the reaction volume down to 2 µL and using as little as 20 pg of input cDNA. The resulting sequencing data were bioinformatically analyzed and correlated among the different library reaction volumes. Our results showed that decreasing the reaction volume did not interfere with the quality or the reproducibility of the sequencing data, and the transcriptional data from the scaled-down libraries allowed us to distinguish between single cells. Thus, we have developed a process to enable efficient and cost-effective high-throughput single-cell transcriptome sequencing.

Microscale vapour diffusion for protein crystallization.

The development of new crystallization platforms via the application of high-throughput technologies has delivered a plethora of crystallization plates suitable for robot-driven and manual setups. However, practically all these plates (except for microfluidic channel chips) are based on a very similar design and well (precipitant):drop (protein) volume ratios. A new type of crystallization plate (microplate) has therefore been developed and tested that still employs the classical vapour-diffusion technique but minimizes the precipitant well volume to 1.2 microl for a 150 nl protein drop setup. This enables a very significant saving on the total bulk of the crystallization screen, hence allowing the application of new, rare and expensive solutions in automated crystallization-screening procedures. Additionally, owing to the very low drop:well volume ratio, the new microplate can significantly accelerate the equilibrium time necessary for crystal nucleation and growth, in many cases shortening the high-throughput crystallization screening process to a few hours.