Assessing library QC in Illumina Single Cell 3' RNA Prep

Library Yield Expectations

Library yield depends on several factors, including sample type, cell/nuclei input, and PCR cycle number used during the Amplify Library step of the Illumina Single Cell (SC) 3' RNA Prep workflow.

Refer to Illumina Single Cell 3' RNA Prep library QC best practices to confirm accurate QC assessment.

Adjust the number of PCR cycles used during the Amplify Library program to optimize for different input quality and concentration. Review the Illumina Single Cell 3' RNA Preparation library sequencing recommendations and confirm the minimum yield required for optimal sequencing performance.

For more information on recommended PCR cycling parameters, refer to Selecting cycle numbers for High and Low RNA sample types with Illumina Single Cell RNA prep kits

Final Library QC Expectations and Assessment Best Practices

The final Illumina SC 3' RNA Prep library is expected to have an average fragment size ranging between 370-550 bp when the region table is set from 200 to 800 bp.

Library distributions and average fragment size may vary based on sample type and experimental conditions. Refer to Representative library QC traces for Illumina Single Cell 3' RNA Prep for additional examples from different sample types.

Figure 1. Representative fragment analysis of an Illumina Single Cell 3' RNA Prep T2 library from a 5,000-cell HEK 293T/NIH 3T3 mixture using a HS D1000 ScreenTape.

If there are adapter peaks remaining in the final library, these can significantly reduce sequencing efficiency. If these are present, Illumina recommends performing an additional 0.8X magnetic bead cleanup. This extra size selection is not recommended if the library prep concentrations are low. See 0.8X bead cleanup to remove adapter dimers from Illumina Single Cell 3’ RNA Prep libraries for more information.

Refer to Section 3.2: Quality Checks of the Illumina Single Cell 3' RNA Prep Training Packet on the Illumina Single Cell Prep Support page (under the Training heading) for information on final library assessment.

For any feedback or questions regarding this article (Illumina Knowledge Article #9527), contact Illumina Technical Support [e-mail].