Assessing cDNA QC in Illumina Single Cell 3’ RNA Prep
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Ensuring that cDNA meets quality expectations during the "Check QC Product" step is critical for obtaining optimal library QC and sequencing results in Illumina Single Cell 3’ RNA Prep.
Refer to Illumina Single Cell 3’ RNA Prep cDNA QC best practices to confirm accurate QC assessment.
cDNA QC Yield Expectations
cDNA yield depends on several factors, including sample type, cell/nuclei loading, and PCR cycle number used during the "Assess cDNA Quality" step.
Adjust the QC Amplification program to optimize for different input quality and concentration.
If cDNA QC yields ≥ 200 ng/µl in T10, T20, or T100 kits (not applicable to T2 kits), reduce the number of PCR cycles in the "Amplify Library" step by 1-2.
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.
cDNA Fragment Analysis Expectations
The cDNA average fragment size should be > 500 bp when the fragment analyzer region table is set from 200 bp to 5000 bp to proceed with Library Prep.
cDNA peaks, distribution, and average fragment size vary based on sample type and experimental condition(s). For example traces from various sample types, refer to Representative cDNA QC traces for Illumina Single Cell 3’ RNA Prep.
Figure 1. Representative human/mouse cell mixture (HEK 293T/NIH 3T3) using a High Sensitivity D5000 ScreenTape.
If the average cDNA average fragment size is <500 bp, it may indicate significant cDNA degradation.
Figure 2. Degraded cDNA QC product from mouse pancreas nuclei that does not pass QC specifications (<500 bp).
Refer to Section 3.2 “Quality Checks” and Section 3.3 “cDNA Degradation” in the Illumina Single Cell 3’ RNA Prep Training Packet on the Illumina Single Cell Prep Support Page for additional guidance on assessing degraded cDNA.
For any feedback or questions regarding this article (Illumina Knowledge Article #9524), contact Illumina Technical Support .
