# Assessing cDNA QC in Illumina Single Cell 3’ RNA Prep

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](https://knowledge.illumina.com/library-preparation/illumina-single-cell-rna-prep/library-preparation-illumina-single-cell-rna-prep-reference_material-list/000009523) 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.](https://knowledge.illumina.com/library-preparation/illumina-single-cell-rna-prep/library-preparation-illumina-single-cell-rna-prep-reference_material-list/000009521)

**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.](https://knowledge.illumina.com/library-preparation/illumina-single-cell-rna-prep/library-preparation-illumina-single-cell-rna-prep-reference_material-list/000009525)

![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-0b73584d01f196101cf672b54701136b716bf772%2Fimage1.png?alt=media)

**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.**

\*\* ![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-2945f456f67684e95cc8fac9ade858d0fdeac095%2Fimage4.png?alt=media)\
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**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.

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