What are the recommended sample suspension quality requirements for scMLPSeq?

Answer:
The primary input materials for scMLPSeq are FFPE-derived nuclei or 1% PFA-fixed cells/nuclei.

Therefore, traditional cell viability metrics are not strict requirements for this workflow. However, stable sample quality and sufficient input quantity remain important to ensure adequate recovery of usable cells or nuclei.

Recommended input amounts for FFPE-derived nuclei

Total nuclei yield after nuclei isolation: Recommended: >200,000 nuclei (optimal: >500,000 nuclei) 

Input for nuclei preprocessing (pre-decrosslinking): Recommended input: 150–500k nuclei (optimal: 350–500k nuclei) 

Remaining nuclei after preprocessing (after pre-decrosslinking, PFA fixation, washing, and resuspension)

• Total nuclei: recommended minimum: ≥40,000 nuclei (optimal: >70,000 nuclei) 
• Nucleus size: 5-40 μm 
• Aggregation rate: <25%
• Nucleation rate: >10%

Recommended input amounts for 1% PFA-fixed cells or nuclei

For 1% PFA-fixed cells or nuclei suspensions: 

• Total nuclei: recommended minimum: ≥40,000 cells/nuclei (optimal: >70,000 cells/nuclei) 
• Cell/nucleus size: 5-40 μm 
• Aggregation rate: <10%
• Nucleation rate: >70%

Recommended input for probe hybridization

• Recommended range: 40,000–80,000 cells/nuclei 
• Optimal range: 50,000–70,000 cells/nuclei 

Recommended input for droplet generation: 1,000–24,000 cells/nuclei per channel

Different stages of the scMLPSeq workflow require different input ranges. Earlier workflow steps typically require higher input amounts to compensate for nuclei loss during FFPE processing and pretreatment procedures. In contrast, droplet generation requires controlled loading concentrations to minimize doublet rates and abnormal droplet loading. 

As a result, “total nuclei yield” and “final loading input” are distinct parameters and should be evaluated separately.