Single-cell sequencing experiments can be carried out on a variety of samples. In this article, I'll give an overview on the different sample types that can be used as well as some of the specifics. But basically from prokaryotes^1,2 to eukaryotes ^3,4 , single-cell studies were done on anything from which we could isolate cells or nuclei.

There are two main categories of samples that we can use: liquid and solid samples. A liquid sample in single-cell research is fluid from which we can isolate cells or nuclei for analysis. It can include ocean water, bacteria liquid cultures, blood, bronchial-alveolar (BAL) fluid, bone marrow, cerebrospinal fluid (CSF), urine, sputum, or saliva. A piece of biological tissue is a solid sample. Surgical biopsies, animal and plant specimens, and organoid are a few examples. The adherent culture cell lines, which I'll refer to as hybrid samples, sit in the middle of these two categories of samples. Independently of the type of sample that we are using, we need to obtain "happy" individual cells in suspension, the input material for single-cell experiments.

In general terms, the sample preparation protocols for liquid samples are composed of two steps: 1) debris removal and 2) cell washing. For the hybrid samples, we need to add an enzymatic dissociation step where the connection between the cells is disturbed. In the case of solid samples, the protocols are more complex and usually longer. They include the same steps as before plus an extra mechanical dissociation step.

Single-cell sequencing companies insist that, ideally, we should have a minimum of 90% cell viability with zero debris and aggregates. While this is true, this is rarely the case in real life. Seventy-five percent of cell viability will be acceptable as 5%–10% of debris or aggregates. These percentages will depend on the sample type and the sample preparation protocol used. A variety of techniques can be used to improve the quality of our samples if necessary. This is not ideal, though, as we are stressing the cells -with these extra steps- and potentially altering the gene expression. Therefore, it is absolutely essential to optimise sample preparation protocols! Optimising sample preparation protocols is important to ensure the accuracy of our results.

Working with nuclei is the valid option when cellular viability is extremely low (e.g., in snap-frozen samples) or debris is difficult to remove (e.g., brain tissue). Here, the recommendation is to work with intact nuclei in suspension with no clumps and debris. Again, in real life, we will have some nuclei clumping together. In this case, the amount of nuclei clumps should not exceed 20%, as more nuclei clumps will affect the experiment's quality. Nuclei will also be required if we are working with solid tissues such as fat tissue or if we want to study large cells (e.g., skeletal muscle cells and cardiomyocytes). Until today, using droplet or micro-, nano-, or pico-well-based methods was not suitable for large cells (> 70 um) since it is difficult to fit them into the capillary or small wells used by this type of technology.

I hope you have enjoyed this blog and gained new insights into the different types of samples we can use for single-cell sequencing. As I always say, the more we know, the better we can plan our experiments.

Thanks for reading!

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REFERENCES:

1. Seeleuthner. (2018). Nature Communications 9 no. 

2. Ehsan, Christina, Antoine-Emmanuel, & Jörg. (2020). Nature Microbiology 5 no. 

3. Sarah, Victoria, Avery, Karla, & David. (2017). The ISME Journal 11 no. 

4. Rong, Jin, Wei, Bo, Jiang, Xianjun, & Si. (2021). Journal of hematology oncology 14 no.