Besides RNA quality, what are the requirements for nuclei count and nuclei suspension quality?

Answer:
In addition to RNA integrity, nuclei quantity and suspension quality strongly influence final spatial data quality.

H&E assessment: Determine the target tissue region and nuclear size to decide slice thickness.

Recommended tissue cellularity

After H&E assessment, the total estimated nuclei number across the tissue area corresponding to one capture area should generally exceed ~100,000 cells. 

Cell numbers may be estimated using CellProfiler (https://cellprofiler.org/) analysis of H&E-stained adjacent sections. 

Insufficient tissue cellularity may result in low captured cell counts, inadequate spatial coverage and reduced downstream spatial analysis quality.

Recommended nuclei suspension QC

• Nucleated particle rate: >5%. Low nucleated particle rates may reduce captured nuclei counts and median gene counts.
• Aggregation rate: ≤20%. High aggregation rates may increase droplet emulsion clogging risk and doublet rates. If aggregation exceeds 20%, filtration using a 20 μm cell strainer is recommended.