Table 3.

Example 250 nM aptamer staining solution

Solution 1
Reagent (concentration)	Final concentration	Volume to add
Biotinylated aptamer (10 μM)	250 nM	2.5 μL
PBS +/+	N/A	2.5 μL
1. Denature aptamer structure by heating for 5 min at 65°C. 2. Allow to fold by cooling at room temperature for 3 min. 3. Add in the reagent below to make Solution 2.

Solution 2
Reagent (concentration)	Final Concentration	Volume to add
Streptavidin dye (5 μM)	125 nM	2.5 μL (0.5 M equivalent compared to aptamer)
1. After adding the streptavidin dye, incubate in the dark at room temperature for 20 min. 2. Add in the reagents listed below to make the final aptamer staining solution, Solution 3 (total volume of Solutions 1 + 2 + 3 = 100 μL).

Solution 3
Reagent (concentration)	Final Concentration	Volume to add
ssDNA (10 mg/mL)	1 mg/mL	10 μL
PBS+ or Media+	N/A	82.5 μL
1. After adding ssDNA and PBS+ or Media+, incubate in the dark at room temperature for 10 min (this ensures that any remaining biotin binding sites on the streptavidin dye are quenched).