> For the complete documentation index, see [llms.txt](https://knowledge.illumina.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://knowledge.illumina.com/library-preparation/illumina-single-cell/library-preparation-illumina-single-cell-reference_material-list/000009394.md).

# Extending cDNA storage time in Illumina Single Cell 3' RNA Prep

**Extending storage time of isolated cDNA beyond 72 hours at -85°C to -65°C after the Clean Up cDNA step during the Illumina Single Cell 3' RNA Prep workflow is not recommended.**

Limited five-cycle PCR during cDNA (ie, whole transcriptome) Amplification results in lower cDNA concentrations. Extended storage may lead to cDNA degradation, increasing the risk of library QC failure during the Check Library step. For optimal results, adhere to the specified storage conditions and duration, and avoid storing isolated cDNA in amplicon-contaminated environments.

**⚠️** Even if cDNA QC results from theCheck QC Product step are not available, proceed with Library Preparation within 72 hours.

While cDNA is sensitive to prolonged storage, final Illumina Single Cell libraries can be reliably stored for up to 30 days at ‑25°C to ‑15°C.

**Additional Recommendations:**

* When working with extremely low cell or nuclei inputs in T2 reactions, Illumina recommends minimizing the amount of time used at this stopping point to achieve optimal sensitivity.
* If there is no immediate access to a fragment analyzer for cDNA QC, amplify and purify the cDNA QC product and store it at -25°C to -15°C for up to four weeks until fragment analysis can be outsourced along with the final Illumina Single Cell libraries.
* Refer to [cDNA QC best practices](/library-preparation/illumina-single-cell/library-preparation-illumina-single-cell-reference_material-list/000009523.md) and [Assessing cDNA QC in Illumina Single Cell 3' RNA Prep](/library-preparation/illumina-single-cell/library-preparation-illumina-single-cell-reference_material-list/000009524.md) for more details on proper cDNA QC product assessment.

**Resolution:**

If [library](/library-preparation/illumina-single-cell/library-preparation-illumina-single-cell-reference_material-list/000009527.md) preparation fails despite obtaining QC-passed cDNA, the Illumina Single Cell Supplemental Enrichment and Amplification Kit (SEA Kit) (20132794) can be used to generate a second batch of cDNA from the retained PIP pellet stored at -80°C for up to three months.

The recovered cDNA will then need to be used with the Illumina Single Cell Library Prep Kit (20132789) to prepare a new sequencing-ready library. This recovery method avoids having to restart from cell loading with a new sample.

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #9394), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000009394%20-%20Library%20Preparation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


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