Top 5 tips for sorting (FACS) upstream of single-cell RNA-seq experiments

Getting high-quality scRNA-seq data depends on the ability to quickly and carefully capture single, live cells to prevent RNA degradation prior to library prep. Successful sorting is tricky, requiring some careful planning and optimization of FACS techniques. Here is a list of tips to help you avoid common problems, including recommendations on collection parameters that maximize sample quality upstream of our most popular library prep kits.

Don't clog the sorter

Nothing is worse than a clogged flow cytometer! It can slow down sorting and compromise your samples. Therefore, you should ensure your single-cell suspension is free of cell debris and clumps by removing them using centrifugation, cell strainers, or other methods. Optimize your dissociation technique to maximize the number of single cells without compromising cell viability. As magnesium and calcium are required by many adhesion molecules, we recommend suspending bulk cell suspensions in cold, filtered 1X PBS (without EDTA, Mg²⁺, and Ca²⁺, and supplemented with 1–2% BSA) or BD FACS Pre-Sort Buffer (Cat. # 563503), which already contains a serum protein that can help maintain viability. Counterintuitively, sorting cells suspended in media can threaten viability (Kissner 2015) and often inhibits cDNA synthesis. We strongly recommend using a viability stain prior to sorting to maximize the capture of live single cells.

Treat the cells gently

FACS is rough on cells, but there are things you can do to minimize cell damage and lysis during sorting. In addition to following the pre-sort buffer guidelines in Tip #1, we recommend using large nozzle sizes and slower flow velocities, especially for fragile cell types. Work with your flow cytometry core facility or instrument manufacturer to find the appropriate settings for your sorter. Finally, sorting rare cell types, especially at low flow velocities, can be time-consuming. Therefore, you may want to consider using a pre-enrichment step before FACS or a two-step sorting strategy.

Sort into the bottom of the wells

This seems straightforward, but it is a common problem. Ensure your flow sorter is calibrated to deposit droplets containing cells into the bottom of the wells of your plate/tube strip(s). If your plate/strip(s) are not aligned appropriately, cells may end up on the side of the wells, where they will quickly dehydrate, risking RNA degradation. Even worse, the cells may not end up in the wells at all. Work with your flow cytometry core facility or instrument manufacturer to calibrate your sorter. You may also want to read up how some researchers have developed quality control methods using fluorescent beads (Zigon et al. 2018), reporters (Rodrigues and Monard 2016), or even Litmus paper (Baumgartner et al. 2014) to ensure droplets are delivered appropriately.

Sort into lysis buffer

Once the cells have been sorted, the transcriptome should be stabilized as quickly as possible. Thus, we recommend sorting into cold, fresh lysis buffer containing RNase inhibitor. Once cells have been deposited into plates/tube strips, they should be gently centrifuged at 100g for 15–30 sec. If not proceeding immediately to cDNA synthesis, place the samples on dry ice to flash-freeze and store at –80°C for up to several weeks. These steps ensure sorted cells are in the lysis buffer (as droplets can sometimes ricochet), where they can lyse in the presence of RNase inhibitor. See the table below for recommendations tailored to our single-cell RNA-seq kits.

Ask for help

FACS is complicated, and every sorter is different. Reach out to your flow cytometry core facility or instrument manufacturer before you start your experiment. These experts can help you get your experiment right.

Happy sorting!

Takara Bio has pioneered and systematically advanced single-cell RNA-seq technology for years, and we are committed to helping advance your research. Please check out our single-cell RNA-seq kits to find the right one for your application, including oligo(dT) and random-priming solutions.

References

Baumgartner, T. et al. "Direct visual confirmation of sort material deposition alignment at the bottom of 96‐ and 384‐well PCR plate wells." Poster presented at the XXIX Congress of the International Society for Advancement of Cytometry, Ft. Lauderdale, FL, May 2014.

Kissner. How Cell Culture Medium Can Decrease Cell Viability During A Flow Cytometry Cell Sorting Experiment. Expert Cytom. (2015). at <https://expertcytometry.com/how-cell-culture-medium-can-decrease-cell-viability-during-a-flow-cytometry-cell-sorting-experiment/>

Rodrigues, O. R. & Monard, S. A rapid method to verify single-cell deposition setup for cell sorters. Cytom. Part A 89, 594–600 (2016).

Zigon, E. S. et al. A rapid single cell sorting verification method using plate-based image cytometry. Cytom. Part A 93, 1060–1065 (2018).