Is RNase inhibitor required for SeekOne single-cell workflows (transcriptome/multiome)? What is the recommended amount?

Answer:
Yes. RNase inhibitor is strongly recommended for all RNA-containing samples, including live-cell and nuclei suspensions. The reagent is not included in the kit and should be prepared by the user.

The recommended final concentration is 1 U/μL.
Validated product: Sigma-Aldrich #3335402001. Equivalent products from other suppliers may also be used after confirming compatibility with reverse transcription reactions.

Recommended usage by workflow:

• Standard scRNA-seq (live cells):
  For RNase-rich tissues such as pancreas, liver, spleen, salivary gland, intestinal mucosa, and some digestive tumors, adding 0.4 U/μL RNase inhibitor to the cell suspension may improve RNA recovery and median gene counts.
• snRNA-seq:
  Strongly recommended to include 1 U/μL RNase inhibitor together with 0.1% BSA in nuclei suspension buffers to reduce nuclei aggregation and nonspecific adsorption.

• ATAC + RNA multiome: RNase inhibitor is mandatory. Based on empirical experience:
  • Frozen tissue nuclei preparation typically requires ~12,000 total units/sample
  • Cell-derived nuclei preparation typically requires ~8,000 total units/sample
  • These values are reference estimates and include total usage across all nuclei isolation buffers. Actual amounts should be adjusted according to sample number and workflow scale.

Operational recommendations:

• Keep RNase inhibitor on ice during use
• Avoid repeated freeze–thaw cycles
• Mix gently after buffer addition; avoid vigorous vortexing

For multi-sample workflows, prepare single-use aliquots to reduce contamination risk