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So you think you want to do a single cell RNA-seq experiment?

What should I do first?

 

Optimize Sample Prep

 

The only way to have a successful single cell experiment is to start with a suspension of high viability individual cells that are free of debris. 

Not sure where to start? Start with a dissociation protocol for flow cytometry or with some established protocols from the single cell literature. A good place to look is the Broad sc/sn-RNA-seq Toolkit paper (https://www.nature.com/articles/s41591-020-0844-1) or the 10x Genomics support page (https://support.10xgenomics.com/single-cell-gene-expression/sample-prep

 

If you’re working with a non mammalian species you may also need to optimize a counting strategy. We typically use AOPI with a Countess II Fluorometer to count cells, but have found it is not an effective method of counting protoplasts.  

 

Send us pictures if you’re not sure if your single cell suspension is clean enough for a chromium experiment.

Common Problems:

Too much debris? Try …

An optiprep gradient

Low speed centrifugation

Myelin Removal Beads (brain tissue)

RBC Removal Beads / lysis step (if debris appears to be RBCs)

Adding a sort step (works well with nuclei and immune cells)

 

Too dead? Try…

Keeping dissociation short and sample handling to a minimum

Dead Cell Depletion by magnetic column

Low speed centrifugation

Sort for viability

 

Too clumpy? Try…

Short treatment with Trypsin

Filter with a Flowmi filter

Add DNase *

Add EDTA *

* DNase and EDTA can be used as part of a dissociation/ sort buffer but will need to be washed out of the final loading buffer for your single cell experiment

 

Worried that some aspect of your sample prep is not compatible with scRNA-seq? Try using your cells as input into a low input RNA-seq kit (available from NEB, Takara or DIY based on the SMART-seq protocol) and proceed through cDNA amplification to make sure you can generate full length cDNA based on a gel/bioanalyzer. For nuclei you can also perform a Trizol RNA extraction and check the RNA quality on the bioanalyzer to make sure RNA degradation is not occurring during your sample prep. 

Figure 5 from Regan & Preall 2022

Images of different types of cell suspensions (Regan & Preall, 2022)

What Next?

Consider Experimental Design

 

  1. Can you get enough cells from one individual? Or will you need to pool multiple individuals to reach ~50000 live, single cells per sample? How does this fit with your workflow?

  2. Do you want all cells from your tissue or will you need to enrich for cells of interest? Consider if you will need to optimize an antibody panel for flow, or use magnetic columns to enrich/deplete certain cell types. Can you still get enough cells after the enrichment?

  3. Is sex a variable in your experiment? If not you’ll want to make sure to balance sex across conditions/treatments

  4. Will the timing of sample collection allow the experiment to be completed during business hours or will some sort of cryopreservation/fixation/other sample storage strategy be required. 

Think About Analysis 

 

  • Not working with Human or Mouse samples or need to add custom genes? Start thinking about what genome you want to use.

  • Who’s going to be analyzing your data? If it’s your first time getting single cell data you might want to familiarize yourself with analysis tools - start with the basics by downloading Loupe Cell Browser or taking a look at the Seurat (R) and/or Scanpy (Python) tutorials

For more information about planning a single cell experiment check out our paper in Current Protocols: https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/cpz1.498 

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