Illumina Single Cell 3’ RNA Prep cDNA QC best practices

Importance of cDNA QC

Ensuring the quality of both the cDNA QC product and the final library is critical for achieving optimal sequencing performance in the Illumina Single Cell 3' RNA Prep workflow. Proper QC assessment ensures that the cDNA is of sufficient quality to generate satisfactory final libraries and verifies that upstream steps, including sample preparation and workflow handling, were performed correctly.

Recommended cDNA QC Best Practices

When determining the average cDNA fragment size during the "Check QC Product" 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

• Perform QC within four weeks if storing cDNA QC product at -25°C to -15°C following the "Clean Up QC Product" step.

• If submitting to a core lab/service provider for fragment analysis, submit the cDNA QC Product with the final libraries. Review the key considerations below for further instructions.

2. cDNA 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 cDNA. Set the region table 200 - 5000 bp for cDNA QC analysis.

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

• TapeStation 1 - 10 ng/µl (HSD5000 ScreenTape)

• BioAnalyzer 1 - 2 ng/µl

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

Below are representative Illumina Single Cell 3’ RNA Prep T10 cDNA QC fragment analysis results from a HEK 293T and mouse NIH 3T3 cell line mixture on a BioAnalyzer HS DNA Assay (top) and TapeStation HSD5000 ScreenTape (bottom) with the appropriate region table assignments from 200 bp to 5000 bp.

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Note: These samples were processed from different cell suspensions during different experiments. Variability in peak shape and distribution may occur between instruments and sample types.

Key considerations for proceeding with Illumina Single Cell Library Prep

• Refer to Assessing cDNA QC in Illumina Single Cell 3’ RNA Prep to determine if cDNA is satisfactory to proceed with Library Prep.

• To ensure high-quality output, proceed with Library Prep within 72 hours of completing the "Clean Up cDNA" step, even if still awaiting fragment analysis results from the cDNA QC product.

• Illumina recommends proceeding with Library Prep on purified cDNA as soon as possible after the "Clean Up cDNA" step for optimal results.

• For further guidance on cDNA storage, refer to Extending cDNA storage time in Illumina Single Cell 3' RNA Prep

⚠️ Do not use the cDNA QC product as input for Illumina Single Cell Library Prep, as this will result in assay failure. Only the total volume of cDNA recovered from the "Clean Up cDNA" step can be used for library preparation.

For any feedback or questions regarding this article (Illumina Knowledge Article #9523), contact Illumina Technical Support [e-mail].