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.

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.

M osquito R from TTP LabTech Ltd. is an innovative nanoliter dispenser that combines the liquid transfer capability of a fixed head pipette with the elimination of cross-contamination, using disposable tips. For many applications required in genomics, proteomics and drug discovery, Mosquito can reduce assay cost by minimizing reagent and sample usage.

The automation of protein crystallography screening has contributed significantly to the rapid progress of crystallographybased structural biology offering increased throughput and accuracy, with the ability to use smaller volumes of both protein and screen solutions, thereby saving valuable protein and reducing reagent costs. Automation of protein crystallisation trials set-up requires accurate placement of nanolitre volumes of protein and screen drops, in addition to the reproducible and accurate dispensing of solutions of varying viscosities. This is particularly important for the set-up of the highly viscous lipid mesophases in the LCP crystallisation technique for membrane protein crystallisation trials.This poster discusses demonstrates the ability of TTP Labtech's mosquito ® Crystal and mosquito ® LCP to address the issues of the set-up of automated protein crystallisation screen trials. The ability to automate both micro batch and vapour diffusion methods of protein crystallography (sitting drop, hanging drop) without instrument configuration change offers significant flexibility for the crystallography laboratory. mosquito LCP offers all the advantages of mosquito Crystal but with the addition of a dedicated microsyringe dispenser for accurate dispensing of nanolitre volumes of the highly viscous cubic phase. mosquito Crystal and mosquito LCP offers fast throughput, high precision and unrivalled reproducibility.

Growing protein-ligand complex crystals can be challenging, especially in cases where the affinity is poor and the solubility of the ligand in the crystallisation condition is low. Various methodologies are often trialled before obtaining a diffraction-quality protein-ligand crystal.Co-crystallisation is a common method for producing protein-ligand complex structures. It is especially useful when drug-like compounds trigger conformational changes in proteins. This can result in variations in the growing conditions or crystal forms, and may necessitate wider screening strategies for co-crystallisation in general.Alternatively, soaking protein crystals with ligands is the fastest route to produce highthroughput structures, as long as the starting crystal form is easy to grow reproducibly, able to accommodate the desired ligand and, is robust to physical and chemical changes.This poster will describe automated low-volume, high-throughput techniques for both types of crystallisation methods.