Expert Insight: Sample preparation and enrichment for single-cell sequencing assays

---

Dr. Nicole Abreu, Science & Technology Advisor, 10x Genomics

Single-cell RNA sequencing (scRNA seq) has emerged as a powerful tool that can reveal complex and rare cell populations, uncover regulatory relationships between genes, and trace connections between cellular lineages in different cell types and rare populations. The high sensitivity of scRNA seq requires careful processing of the sample to ensure optimal results.

In this SelectScience^® webinar, now available on demand, Dr. Nicole Abreu, Science & Technology Advisor at 10x Genomics, explores the basics of sample preparation, sample cleanup and enrichment methods to obtain quality data from single-cell sequencing assays. Additionally, understand how to leverage 10x Genomics multiplexed approaches to profile gene expression, antigen-binding specificities and classify immune cell types.

Read on for the highlights of the live Q&A session or register to watch the webinar at a time that suits you.

Q: How many cells/events can the immune-profiling method collect?

NA: The range for our products is typically anywhere from targeting 500 to 10,000 cells per reaction. If you wanted to go larger than that, you can use multiple reactions and aggregate the data afterwards. We do have data sets online that have a million cells in them, where multiple reactions were used and the data combined. 

Q: In the immune-profiling workflow, can the cells be recovered, or is the workflow destructive? 

NA: Since the cells must go through the microfluidic chip to generate the single-cell GEMs for analysis, they do become lysed in that process. You can do the reverse transcription and generate the single-cell gene-expression profiles. Since the cells are lysed in the GEMs, you are getting the gene-expression profile of the entire cell. 

Q: Are gene expression profiles significantly different between fresh and frozen samples? 

NA: In prior publications where we have tested fresh versus frozen, using Peripheral Blood Mononuclear Cells (PBMC), results showed there was a very high similarity between their average gene-expression profiles. From that perspective, sample type has very little impact on the cryopreservation. However, different sample types may have different susceptibilities to freezing. 

Q: What is the recommended cell viability cut-off before I consider cleaning up my sample?

NA: It would depend on the type of sample you are working with. The higher the viability, the better. We’ve looked at PBMCs and blood cells and 90% viability or higher is what we commonly recommend, with those types of cells or cells in culture that are amenable to the trypsinization and single-cell generation, suspension generation techniques.

However, there are sample types like tumors that have necrotic tissue. It’s likely that these sample types will have more dead or dying cells. I can't give a strict cut-off, but there are dead-cell removal kits that you could use to increase your viability percentage. When you're making those decisions, people like myself can help because adding additional steps of manipulation would increase the length of time where you're processing those samples. I would say it depends on the sample type. 

Q: During the upstream process, can you add the barcode to the sonicated DNA in the cell/nuclei? Is there any specific size bp of DNA required for indexing?

NA: That would be some form of custom application. In the case of gene expression where we're looking at the RNA molecules of the individual cells, adding the barcode to sonicated DNA in the cell or nuclei would mean that you're lysing your cells during the sonication process. That might be more of a bulk approach. 

Q: For feature barcoding, is there a maximum number of protein markers you can detect using 10x?

NA: Each of the individual types of antibody will have a unique barcode associated with it. The upper end is limitless when it comes to the number of antibodies. In the real world, when you design these pools and panels of antibodies, you want to consider the titration of the antibodies when you're doing the staining methods.

There are some pre-titrated panels that are available. One example, for TotalSeq-C, is produced by BioLegend, one of our compatible partners. They have a pre-titrated panel of about 120/130 different human immune markers. That’s one example where you can go above 100 and I know of publications where people have gone higher.

Q: Can I use feature barcoding on tissue samples?                                                                     

NA: Yes. We have protocols on our website that go over antibody staining, for example, the consideration to dissociate that tissue first to generate a single-cell suspension. Then you can use that as your input for the antibody staining process.

Q: Are all the products focused on eukaryotic cells?

NA: Many of our products have been designed to capture off the poly(A) tail of mRNAs, so that does limit it to eukaryotic organisms. We have had some examples where customers have spiked, using custom primers, to pick up viral transcripts in our immune profiling assays. That’s a potential way to kind of hack the system and study virus in addition to the immune system.

Q: Do you provide or recommend analysis platforms/software?

NA: Yes, we have cell ranger analysis pipelines that are included with our solutions. We've got plenty of information about those on our support website. I also help advise people when it comes to what direction they want to go, from a data analysis perspective.

As far as third-party tools, many researchers choose to use tools such as Seurat and third-party analysis to generate graphs for their publications. It’s dependent on the capabilities and resources that the researcher has to run the analysis.

SelectScience runs 10+ webinars a month across various scientific topics, discover more of our upcoming webinars>>