PIXUL: Developing a High-Throughput Brain Single Nuclei Isolation Protocol for Gene Sequencing Applications

Abstract

Background For improved gene sequencing efficiency, in particular snRNA-seq, there’s a growing need to develop a protocol capable of isolating nuclei from multiple biological tissue samples at a high-throughput rate while maintaining cost efficiency. Traditional, manual single-nuclei isolation protocols require tissue samples to be individually homogenized using a Dounce homogenizer or a pestle, which limits the number of biological samples that can be processed in a single isolation run. The PIXUL multi-sample megasonicator offers a potential alternative approach to manual homogenization by using ultrasound sonication to automate the homogenization of up to 96 tissue samples in a single isolation cycle.

Methods This project investigates the potential implementation of the PIXUL to homogenize multiple brain tissue samples and generate enough nuclei for gene sequencing applications at a cost-efficient, consistent, high-throughput rate. To test its capabilities of consistently homogenizing multiple brain tissue samples, a single-nuclei isolation protocol integrating the PIXUL was designed, tested, and refined based on preliminary experimental results. Tissue lysis and nuclei quality were assessed using trypan blue staining and microscope imaging. Resulting nuclei concentrations were calculated using a manual hemocytometer counting chamber. ResultsThe protocol yielded nuclei concentrations between 9.35 x 105 and 2.01 x 106 nuclei/mL, which is a comparable range achieved by other manual and automated single nuclei isolation protocols. However, a significant amount of cellular and nuclei debris was observed in all replicates after samples were homogenized. The presence of this debris is likely due to extended sample suspension in lysis buffer, as well as the presence of myelin. This debris may interfere with high-throughput gene sequencing applications. Conclusions Although this protocol was able to achieve nuclei concentration values that were comparable to other isolation methods for all of its replicates, further experiments focusing on optimizing the PIXUL’s setting, finding a method to removal myelin and extracellular DNA, and count nuclei at a high-throughput rate are necessary to improving the reproducibility and efficiency of this novel, high-throughput brain single nuclei isolation protocol.