# Best practices for cell preparation for Illumina Single Cell prep

Sample quality is critical for optimal results with Illumina Single Cell 3' RNA Prep. Ideal cell suspensions are 90% viable or higher, and contain minimal debris or aggregates.

The best option is to start with fresh cells whenever possible, and nuclei sequencing is required when starting from frozen tissue.

If cells are handled too roughly, cells will lyse which can increase background mRNA. To minimize damage during sample preparation, pipetting and centrifugation should be kept to a minimum. Tightly packed cell pellets require extra pipetting, which can damage cells from shearing effects. Pipetting steps should be slow and gentle, and the use of wide-bore tips is recommended to minimize shear forces on the cell suspension.

It can be necessary to use standard-bore tips in some cell/sample types to reduce clumps.

**Figure 1**: Common Cell Preparation Reagents that are Incompatible with Illumina Single Cell

![](/files/LhufrbJqZns6NI5eDjXh)

It is critical to complete the Cell Suspension Buffer Wash of cell suspensions prior to input into the PIPs to ensure any inhibitory reagents are sufficiently washed out of the cell suspension.

DNase I can break down the cell barcode structure and PolyA capture moiety on PIPs. If DNase I must be used, make sure it is thoroughly washed out of the cell suspension by doing at least three 2 ml PBS washes followed by a 1ml Illumina Single Cell Suspension Buffer wash.

User supplied RNase Inhibitor (0.4-1U/μl) may need to be added to any buffers during cell preparation of time-consuming steps (eg, antibody staining buffers, FACS collection tube buffers, and the final cell suspension during cell counting and viability checking).

Including RNase Inhibitors in upstream processes is especially important for challenging sample types with high endogenous RNase and/or low mRNA content (eg, neutrophiles, eosinophils, adipose, pancreas, spleen, machrophages, etc.). It is usually not necessary to add RNase inhibitors for washing steps and washing buffers unless working with granulocytes.

Illumina Single Cell 3’ RNA prep kits provide enough RNase Inhibitor to add to samples into the PIP tube during the Capture and Lysis portion of the workflow.

**If there is an expected delay between completing cell counting and viability assessment, and starting the Capture and Lysis step, RNase Inhibitors should be added directly into the diluted cell suspension instead of spiked in separately into the PIP tube during sample addition.**

For more information refer to Section 2 in the Illumina Single Cell 3’ RNA Prep [Training Packet](https://support.illumina.com/sequencing/sequencing_kits/illumina-single-cell-prep/training.html).

\
\
\ <br>

|                                                                                                                                                                                                                                                                                                                                                                              |
| :--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------: |
| *For any feedback or questions regarding this article (Illumina Knowledge Article #9437), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000009437%20-%20Library%20Preparation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


---

# Agent Instructions: Querying This Documentation

If you need additional information that is not directly available in this page, you can query the documentation dynamically by asking a question.

Perform an HTTP GET request on the current page URL with the `ask` query parameter:

```
GET https://knowledge.illumina.com/library-preparation/illumina-single-cell-rna-prep/library-preparation-illumina-single-cell-rna-prep-reference_material-list/000009437.md?ask=<question>
```

The question should be specific, self-contained, and written in natural language.
The response will contain a direct answer to the question and relevant excerpts and sources from the documentation.

Use this mechanism when the answer is not explicitly present in the current page, you need clarification or additional context, or you want to retrieve related documentation sections.