Tabula Muris, a single-cell Atlas of the adult mouse

In a SelectScience webinar, now available on demand, Spyros Darmanis, Cell Atlas Group Leader at the Chan Zuckerberg Biohub, presents a compendium of single-cell transcriptomic data from the model organism Mus musculus, which comprises more than 100,000 cells from 20 organs and tissues.

This exciting data represents a new resource for cell biologists, revealing new insights into gene expression within poorly characterized cell populations. It also enables the direct and controlled comparison of gene expression in cell types that are shared between tissues, such as T lymphocytes and endothelial cells.

Watch this webinar to learn about:

• Single-cell RNA-seq technologies and their application in a large-scale Cell Atlas project
• The advantages of combining shallow droplet methods with highly sensitive plate-based Smart-seq2
• How the latest liquid handling technologies enable cost-effective, miniaturized NGS workflows with flow-sorted cells

Think you could benefit from this webinar, but missed it? You can now watch it on demand at a time that suits you and find highlights from the live Q&A session below>>

Q: What is your current cost per cell in the plate process?

SD: The unit cost right now is around $500 per plate and every plate will yield a maximum of 360 or so cells. So, this works out a little over $1 per cell. Of course, this depends a lot on your ability to sort cells, so we prefer to calculate by plate. For example, if you can only recover a single cell from a plate, then the cost for that cell would be $500.

Q: What is the typical doublet rate with the droplet method and with fluorescence-activated cell sorting (FACS)?

SD: The droplet doublet rates are calculated by the manufacturer and there's a table in the manual that you can consult that tells you, for whatever number of cells you're trying to recover, what your estimated doublet rate is. What we usually try to do is keep the doublet rate below 7%. So, our usual doublet rate for droplets is between, 3% and 7%. However, for FACS, your doublet rate will depend on your ability to gate and sort single cells. Assuming you are doing everything perfectly, your dissociation works, and you have the proper doublet gating on your FACS, then I would estimate this to be in the same order as the droplets for most cases.

Q: Do you do a bead clean-up of the cDNA before library prep? And if not, how do you avoid that step?

SD: It is possible, but it doesn't align with the throughput we're trying to achieve here, and it increases the cost dramatically. We found that a dilution of the cDNA helps and we also saw that Tn5, the enzyme we use for library prep, is not inhibited. So, we don't do any bead clean-up on the cDNA before the library prep, but we do a bead clean-up on the combined libraries at the end and before they get loaded in a sequencer.

Q: How many plates per day can you process?

SD: With the current setup we have, we can do around 15 plates a day (for the post-cDNA, library prep) and around maybe 20-22 plates a day for the cDNA prep.

Q: Is it possible to have a Cell Atlas for humans rather than for a mouse?

SD: Yes, there are already efforts, one being the Human Cell Atlas consortium. With that, and with efforts from others, we will soon have a Cell Atlas of the human. There are also existing Cell Atlases of individual organs from humans.

Q: Why do the three platforms detect quite distinct gene sets from single cells in the Venn diagrams?

SD: To clarify, what's showing in the Venn diagram is not the detected genes. Instead, it is the list of differential expressed genes between that cell type and any other cell type within that tissue. The three different methods are reporting beyond the core of the yellow genes and are reporting an additional number of genes for that cell type that other methods do not. There is the element of sensitivity, as some methods like the FACS will be detecting more genes. However, you also need to factor in noise, as many of these differential expressed genes are probably just artefacts of the procedures. So, the difference can be accounted for by method and sensitivity. Additionally, if the gene is detected, the process by which we recover these differential expressed genes is not error-free.

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