Quality and validity of results derived from single-cell genomics (SCG) and other next-generation sequencing (NGS) technologies strongly depends on an adequate upstream sample preparation. Learn how to get optimal sequencing results with the help of gentle tissue dissociation and cell sorting protocols.
Single cell genomics and other next-generation sequencing applications depend strongly on adequate upstream sample preparation. Results can easily be affected by poor cellular viabilities, the presence of non-target cells or debris, or by a high contaminating background of free DNA or RNA. In addition, harsh conditions during sample preparation workflows can easily cause alterations to transcriptome, and thereby generate unwanted artifacts. This is especially troublesome if you are interested in genes related to cellular stress, activation, or metabolism.
Unlike conventional droplet sorters, the MACSQuant Tyto Cell Sorter does not subject cells to excessive pressure, strong decompression, high shear forces, or exposure to electrical charge. By employing a gentle microchip, this cell sorting approach results in the highest cellular viabilities without exposing cells to stressful conditions. Furthermore, the MACSQuant Tyto Cell Sorter is equipped with a temperature-controlled cooling unit, stabilizing your samples at a temperature of your choice from 4–25 °C.
Coming from the input chamber, cells enter the microchip through a microchannel where they are interrogated by three lasers. Before entering the microchannel, potential cell aggregates are held back by a filter system, guaranteeing a smooth sorting process. When a target cell (magenta) is identified, a magnetic pulse coming from the solenoid opens the microvalve, which then redirects the target cell into the positive collection chamber. In the default state, the valve is closed allowing non-selected cells (blue and orange) to flow through into the negative collection chamber.
Data provided by Quy Nguyen (Kessenbrock Lab, University of California, Irvine) demonstrate lower impact on EpCAM+/CD49f+ murine mammary gland epithelial cells sorted with the MACSQuant Tyto compared to conventional cell sorters. Prior to sequencing, library prep was performed using the 10x Chromium® system.
Side-by-side comparison of gene expression of cells sorted on the MACSQuant Tyto and on a conventional droplet-based cell sorter. Dots colored in red indicate which dataset shows higher gene activation. The dot size indicates a relative gene-expression level.
Whole transcriptome (mRNA) analysis showed that cells sorted using the MACSQuant Tyto resulted in higher numbers of total detected genes and unique molecular identifiers (UMIs) compared to cells sorted on a conventional droplet sorter, indicating a high sample quality.
A matrigel sphere formation assay was performed after cells were sorted using either a droplet-based cell sorter or the MACSQuant Tyto. Seven days after seeding, the Tyto-sorted samples showed a significantly higher sphere formation, speaking to a higher viability and functionality of these cells, and matching the results observed in the scRNAseq experiments.