Sample Enrichment Best Practices for Illumina Single Cell 3' RNA Prep

Fluorescence-activated cell or nuclei sorting (FACS or FANS) and magnetic-activated cell sorting (MACS) can be beneficial for the enrichment of target cell populations and can facilitate the exclusion of dead or damaged cells. Enrichment steps also add significant time and can stress the cells, causing an overall decrease in cell health. It can be necessary to perform a second live/dead cell sort following cell sorting to ensure sufficient cell viability.

Dead Cell Removal Kits

It is not recommended to move forward with cells that are less than 75% viable. Adding additional cell wash steps is best if only minor viability improvement is needed. If significant improvement is required, it is recommended to either optimize sample preparation processes and reduce sample preparation time, or to incorporate a Dead Cell Removal Kit (eg Akadeum Life Sciences, Miltenyi Biotec). These kits require starting with a high number of cells (500,000 minimum cells for Akadeum and 1 million cells for Miltenyi).

FACS and FANS

It is common for Fetal Bovine Serum (FBS) to be used in pre-sorting buffers and in collection buffers. As an alternative to FBS (which is inhibitory), Bovine Serum Albumin (BSA) can be used in buffers and for tube blocking. It is critical to complete the cell or nuclei wash step according to the user guide after sorting if the sorted samples contain any potentially inhibitory reagents such as FBS.

If working with low cell or nuclei numbers and there will be no inhibitory reagents in the final suspension, a low volume of Cell Suspension Buffer or 1X Nuclei Suspension Buffer may be used in the collection tube (200-400 μl). Be sure to include the recommended final concentrations of RNase inhibitor and BSA for the sample type being worked with as described in this section. The 1-2 ml wash step may be eliminated if there are no potential inhibitors. Do a post-enrichment count and determine the count for the entire tube. The cells or nuclei may then be concentrated by centrifugation to pellet the samples, then removing a safe volume of the supernatant so as not to disturb the cell or nuclei pellet. Make sure there is sufficient volume of supernatant remaining in the tube to load into the PIP tube for the kit size being used. Resuspend the samples and determine the new count by adjusting the tube count by the new final volume.

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