Why is customization important?
The development of high-throughput single-cell techniques, including commercial solutions, over the last five years has made the analysis of thousands of individual cells in parallel possible. A popular application in the field of single-cell research is the analysis of individual transcriptomes, also called single-cell RNA-Seq (scRNA-Seq). Thanks to this method, researchers are able to study gene expression patterns across a variety of samples to identify cell types, cell states and cell heterogeneity.
One of the methods that was chosen to achieve high-throughput sample processing on commercial platforms is the use of microfluidics techniques. For example, scRNA-Seq on such platforms involves the capture of single cells in microfluidic droplets along with barcoded mRNA capture beads. Commercially available instruments tend to focus on commonly used cell types, i.e. mammalian cell types, with predefined consumables and protocols. However, cells obtained from plants and other less typical organisms may require protocol adjustments of the single cell capture step. This is due to variability in cell size, buffer compositions and other variations. Versatile technology is therefore required in order to successfully apply the capabilities of single-cell research to non-mammalian organisms. Additionally, scientists working on canonical cell types may also wish to develop new protocols to fit with their specific experimental needs. In this case flexible technology would also be of value.
How to customize single cell research
It is possible to customize your single cell research through a range of interchangeable parameters. These parameters (Figure 1) can be adjusted during droplet formation to suit a specific application.
Figure 1: Option to adjust four different parameters during droplet formation on the Nadia Innovate to suit a specific single-cell application.
Temperature control is essential in many biological applications, and requirements vary depending on experimental needs. For example, cold temperature helps maintain a cell’s transcriptome state while heating is required to enable the production of agarose-based hydrogel beads by keeping the agarose molten.
Stirring of cells and beads can be controlled individually on the Nadia Innovate and is used to keep them evenly in suspension. Preventing cells and beads from settling is indeed important to reduce the probability of encapsulating two cells or two beads together. Stirring speeds can be changed depending on the cells’ and/or beads’ density and properties.
Timing, just like temperature control, is important in biology applications. The duration of various protocol steps, such as incubation periods and stirring time, can be adjusted on the Nadia Innovate. For example, a post-run incubation step may be required for improved mRNA capture from cells that are difficult to lyse.
The Nadia Innovate uses positive pressure to push liquids into microfluidic channels and generate droplets. At a set pressure, fluids of different viscosities travel at different speeds and this influences droplet formation. Cells for which standard buffers such as PBS is not recommended, e.g. plant protoplasts, require the user to adjust pressures to achieve stable droplet formation. Added to this, changing pressures also enables researchers to alter the droplet diameter to suit a variety of cell sizes.
The above four parameters can be adjusted in a dynamic manner during a run using the Flow Control Centre software, which controls the Nadia Innovate (Figure 2).
Figure 2: The Flow Control Centre software, which controls the Nadia Innovate platform, enables: (1) real-time visualization of the microfluidic junction, where droplets are formed, and (2) adjustment of parameters during a run for dynamic protocol optimisation.
Get started with the new Nadia Innovate guide
Developing a whole new protocol from scratch can be daunting, especially when one is not familiar with the basic principles of microfluidics. To help make this easier and available to all biologists Dolomite Bio created a ‘Get Started’ guide that walks researchers easily through every stage of protocol optimisation. This new document includes a workflow that guides the user in a step-wise manner through protocol development and single cell customization.