High-quality sample prep is key to a robust ATAC-seq assay. Ensure that your sample preparation is successful both at the initial cell harvesting and nuclei extraction stages before each experiment:
Initial cell harvest (Figure 1A):
While fresh cells are ideal for ATAC-seq reactions, properly cryopreserved cells or nuclei are compatible with the CUTANA ATAC-seq kit workflow as well. Do NOT use flash frozen cells or nuclei as starting material for ATAC-seq.
Cells should be bright white under Trypan blue staining (high Trypan blue exclusion; see red circle for a dead cell staining blue) and in a single-cell suspension before proceeding with nuclei extraction to enable accurate counting. If severe cell clumping is observed, consider the following methods to rescue:
Gentle enzymatic dissociation (if adhesion is strong). If the cells were dissociated from tissue or an adherent culture, residual adhesion proteins may be intact. Treat briefly with a non-enzymatic dissociation buffer (e.g., CellStripper or Versene) or a low-concentration, gentle enzyme like Accutase or TrypLE for 2–3 minutes at room temperature, then quench immediately with media containing FBS or BSA, and gently triturate with a narrow-bore pipette tip.
Gently pipetting. Use a standard P200 pipette to pipette the suspension up and down 10–15 times. The fluid shear force can break physical cell-cell junctions.
Pass the suspension through a 20–30 µm mesh strainer. This physically retains clumps while allowing single live cells to pass through (note: clumped cells will be lost).
Fresh cells will ideally show >90% viability, and properly cryopreserved cells are expected to show >85% viability immediately post-thaw. Optimize cell culture conditions if starting cells have poor viability or appear contaminated.
Nuclei extraction (Figure 1B – shown with some cell debris):
Extracted nuclei should appear >95% “dead” (Trypan blue stained).
Nuclei should have clear borders and be unclumped (visibly <25% aggregation). If severe nuclei clumping is observed, consider optimizing cell harvesting as above (if starting cells were clumping), or optimize lysis time to achieve ideal extraction efficiency with minimal clumping.
Some cell debris after nuclei extraction can be present without negatively impacting downstream data quality.
If possible, aim for 50k human nuclei per standard ATAC-seq reaction.
Figure 1. A) Healthy cells exclude Trypan blue and appear bright white with intact borders. Red arrow, circle: a dead cell or nuclei which has taken up Trypan blue. B) Healthy nuclei take up Trypan blue. They are relatively uniform in size and shape and have intact borders. Cell debris also stain blue and are present among the nuclei.