10x Genomics solutions may or may not be suitable for your project, depending on your sample type and the number of cells. Here, we’ll explain how you should consider these two factors.
As explained in our post What is 10x Genomics? the 10x Genomics Chromium Controller is a microfluidics-based system. In it, the cells of your sample are pushed through tiny tubes, and, importantly, these tubes have a maximum size. This unfortunately means some cells are too large for the microtubes and consequently get stuck. If you are working with large cells, you might want to reconsider using 10x Genomics.
With in-house experiments, researchers at 10x Genomics have determined that 30 µm is the practical maximum. However, the theoretical limit is 50–60 µm.
It is important to note that this maximum size considers cells in a single-cell suspension. Cell sizes in culture or in suspension can sometimes be very different. For example, adhesive neuronal cells can be 100 µm, but in suspension, they shrink so much that they do fit through the tubes.
An example of cells too large to fit through the tubes are cardiomyocytes. Multiple research groups have tried to make the 10x Genomics platform compatible with these heart muscle cells with varying success rates. For example, cardiomyocytes of embryonic mice have successfully been processed, whereas those of older mice clog the system. So, at Single Cell Discoveries, we recommend SORT-seq over 10x Genomics on cardiomyocytes. However, Single Nuclei RNA sequencing is also a possibility.
Another example of a challenging sample type, in general, is neutrophils. Neutrophils have a very low amount of RNA and a high amount of RNases and inhibitors. So, 10x Genomics advises processing these samples fresh, among other additional steps. At Single Cell Discoveries, we could handle fresh samples if you are located close to our lab. Please book a call if this is your case.
Number of cells
We recommend 10x Genomics for projects that aim at sequencing 3,000–10,000 cells. However, you need to send us a lot more cells than 10,000 for various reasons. First, the Chromium Controller captures around 50 percent of loaded cells. Second, many cells are inevitably lost during washing steps. Hence we recommend at least ten million cells per sample. If this is not possible, our minimum required amount is 50,000 cells per sample in duplicate, i.e., you must collect at least 100,000 cells.
If you cannot collect the minimum required amount, are there any options? Yes, thankfully, there are. Although we do not recommend a 10x Genomics project with low cell numbers, we offer other platforms, such as SORT-seq, that can handle fewer cells.
How do I choose?
The answer to this question is simple: check the publications in your field.
We always recommend reading single-cell sequencing papers within your field. This does not only help you with the setup of your experiment, but it can also help you answer your question if 10x Genomics is suitable for your project or not.
We collect all publications from projects that utilized our services here, which you can filter on species, tissue type, and other tags. 10x Genomics itself has an even bigger database here focussed on publications that utilize 10x Genomics.
Regarding cell size, one way to determine if a new or unusual cell type is compatible with 10x Genomics is to test the cells using the Training Kit. The Training Kit is the same as the common Single Cell Reagent Kit except that the training Gel Beads do not include lysis buffer. This means that the partitioned cells are expected to remain intact during GEM generation. You can then examine the GEMs by microscopy for intact cells.
Struggling with your sample type?
Reach out or book a free call to discuss with us.