Cell input and volume loading recommendations for Illumina Single Cell 3’ RNA Prep

Cell and Nuclei Loading Parameters

Cell and nuclei loading in Illumina Single Cell 3’ RNA Prep kits is optimized to balance high recovery rates and data quality. The table below summarizes optimal loading parameters by kit size.

Kit Size	Optimal Cell or Nuclei Input*	Expected Capture Rate	Cell Loading Volume (µl)	RNase Inhibitor Volume (µl)	Target Concentration
T2	5,000	≥ 2,000	4	1	1,250 live cells/µl
T10	17,000	≥ 10,000	5	1	3,400 live cells/µl
T20	40,000	≥ 20,000	8	1 - 2	5,000 live cells/µl
T100	200,000	≥ 100,000	20	4	10,000 live cells/µl

*Exceeding the recommended input increases the risk of a multiplet rate above 8%.

Notes:

• Always include RNase inhibitor in the total loading volume.

• Refer to reference guides for loading volumes to ensure optimal capture efficiency.

Cell and Nuclei Loading Volume Considerations

• Adjust the target concentration if increasing or decreasing input.

• The total loading volume, including RNase inhibitor, must not exceed the specified limit for each kit size. Exceeding recommended volumes may reduce capture efficiency.

• If cells or nuclei cannot be concentrated to the target level, a lower concentration is generally acceptable as long as the total volume remains within recommended limits.

For any feedback or questions regarding this article (Illumina Knowledge Article #9473), contact Illumina Technical Support [e-mail].