Cell sorting tips for 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. Few tips below:

Clean sorter so that no carryover

Clean your sorter with the bleach and distilled water thoroughly before your procedure to avoid any clumps deposit or carry over.

Cell should in single cell suspension to prevent clogging

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. 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, we recommend using large nozzle sizes and slower flow rate, especially for fragile cell types.

Sort into the bottom of the wells

Ensure your flow sorter is calibrated to deposit droplets containing cells into the bottom of the wells plate or tube. If aligned is not appropriate, cells may end up on the side of the wells, where they will quickly dehydrate, risking RNA degradation.

Sort directly in Trizol

Sort cell directly in Trizol, may minimalize post sort handling and you will get a better yield.

Reference:

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).