The protocol recommends ≤15k nuclei input in a 15 μL Fragmentation & Tagging reaction. How should the reaction be adjusted if nuclei input is below or above this range?

Answer:
The core principle is that reagent ratios must remain strictly consistent with the protocol. Enzyme and buffer proportions should not be arbitrarily changed.

Recommended nuclei concentration before fragmentation:

• Approximately 1,700–2,300 nuclei/μL 
• Loading approximately 6.5–8.8 μL nuclei suspension yields ≤15k nuclei in a final 15 μL reaction 

After fragmentation, the total reaction typically contains ≤15k nuclei in 15 μL, corresponding to approximately ≤1,000 nuclei/μL. During droplet generation, 15 μL fragmented nuclei suspension is loaded directly.

If nuclei input is ≤15k

For example, if only 10k nuclei are available:

• Keep the total reaction volume at 15 μL 
• Do not proportionally reduce enzyme or buffer volumes 

Maintaining the standard reaction composition is important for preserving optimal transposase activity and buffer conditions. Lower nuclei input with unchanged reaction chemistry is generally safe and validated.

If nuclei input exceeds 15k

The entire reaction volume must be scaled proportionally.

Examples:

• 30k nuclei → 30 μL total reaction volume 
• 45k nuclei → 45 μL total reaction volume 

All components, including enzyme, buffer, water, and nuclei suspension, should be scaled proportionally.

The kit supports up to a 3× scale-up reaction (45k nuclei in 45 μL total volume) based on the provided reagent amounts.

Backup strategy recommendation

If sufficient nuclei are available, preparing a 2× or 3× scaled fragmentation reaction is recommended. This strategy provides additional fragmented nuclei suspension that can be immediately used for repeat droplet generation attempts if needed, without repeating the entire fragmentation & tagging step.

For example:

• First droplet generation attempt fails 
• Remaining fragmented nuclei suspension can be directly reused for a second loading attempt 

This approach can substantially reduce experimental delay and sample handling time.

Important note:
Regardless of fragmentation reaction scale, the final droplet generation step always uses only 15 μL fragmented nuclei suspension containing ≤15k nuclei per loading reaction. Scaling up the fragmentation reaction provides backup material rather than increasing nuclei input per droplet generation reaction.