Assessing library QC in Illumina Single Cell 3’ RNA Prep
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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.
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 High and Low RNA Sample types and choosing the number of cDNA QC and Amplify Library cycle numbers in 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 large 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. Contact Illumina Technical Support for additional 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 for information on final library assessment. Always check the Illumina support site for the latest version.
For any feedback or questions regarding this article (Illumina Knowledge Article #9527), contact Illumina Technical Support techsupport@illumina.com.
