Illumina Single Cell 3’ RNA Prep library QC best practices

Importance of Library QC

Running through the Illumina Single Cell 3’ RNA Prep workflow and verifying that both the cDNA and final library pass both QC steps is essential for producing quality sequencing results.

Accurate library quantification is necessary for uniform sample pooling and achieving optimal cluster density during sequencing. Review Best Practices for Library Quantification to ensure proper QC assessment.

Illumina recommends proceeding with Library Prep only after confirming the quality of cDNA from the "Check QC Product" step when feasible. Refer to Assessing cDNA QC in Illumina Single Cell 3’ RNA Prep for additional guidance.

Recommended Library QC Best Practices

When determining the average library fragment size during the "Check Library" step of the Illumina Single Cell 3’ RNA Prep workflow, perform the following for consistent and accurate assessment across samples and experiments.

1. Timing of QC Assessment

For optimal results, perform quality checks, pooling, and sequencing of purified Illumina Single Cell 3' RNA Prep libraries stored at -25°C to -15°C within 30 days.

If submitting to a core lab for fragment analysis, submit the final libraries with the cDNA QC product.

2. Library Quantification

Use a Qubit High Sensitivity kit to quantify (in ng/µl) 2 µl of each sample.

⚠️ Do not use the BioAnalyzer or TapeStation to assess concentration.

3. Fragment Analysis

Use an Agilent BioAnalyzer or TapeStation to determine the average fragment size of the library. Set the region table 200 - 800 bp for library QC analysis.

If necessary, dilute samples to ensure they are within the appropriate range of the device.

• TapeStation 1 - 10 ng/µl (HSD1000 ScreenTape)

• BioAnalyzer 1 - 2 ng/µl

⚠️ Loading less than 1 ng/µl on these instruments may not give accurate results.

Refer to Assessing library QC in Illumina Single Cell 3’ RNA Prep to determine if the library is satisfactory to proceed with sequencing.

For any feedback or questions regarding this article (Illumina Knowledge Article #9526), contact Illumina Technical Support [e-mail].