# Best Practices for Ribo Zero Depletion using the TruSeq Stranded Total RNA Protocol

The following describe best practices for Ribo-Zero depletion when using the TruSeq Stranded Total RNA Protocol. Note comments in bold.

**Step 4 of the Bind rRNA procedure found in the** [**Truseq Stranded Total RNA Reference Guide**](https://support.illumina.com/downloads/truseq-stranded-total-rna-reference-guide-1000000040499.html)**, p12**:\
3\. Add 5 µL of the appropriate removal mix to each well, depending on the workflow used.

* Globin removal mix
* rRNA removal mix
* rRNA removal mix Gold
* rRNA removal mix Plant

4. Gently pipette the entire volume of each well of the BRP plate up and down 6 times to mix thoroughly. **It is critical to mix thoroughly**.

**Make RRP**

1. Vortex the room temperature rRNA Removal Bead tube vigorously to resuspend the beads. **The beads must be completely resuspended before addition to the plate**.
2. Add 35 µl rRNA Removal Beads to each well of the new 96-well 0.3 ml PCR plate labeled with the RRP barcode.\
   **Note**:It is important not to skip this step by adding beads to the sample in the BRP plate. Adding the sample from the BRP plate to beads in the RRP plate in step 3 will ensure optimal performance. **It is required to do this even if though it means more steps**.\
   \&#xNAN;**…**
3. Adjust the pipette to 45 µl, then with the tip of the pipette at the bottom of the well, pipette quickly up and down 20 times to mix thoroughly.\
   **Note**:It is important to pipette up and down quickly to ensure thorough mixing. Insufficient mixing leads to lower levels of rRNA depletion.\
   Pipetting with the tip at the bottom of the well and not pipetting the entire volume of the solution helps prevent the solution from foaming. Excessive foaming leads to sample loss, because the foam is not transferred out of the plate efficiently.

**Other points**:\
-Accidentally taking some beads into the RNAClean reaction will add back rRNA into the sample. The indicated transfers may not be sufficient if care is not taken to completely remove the beads.\
-**Never freeze rRNA Removal Beads**.\
\&#xNAN;**-It is important to follow the protocol (including all Notes) step-by-step**.

\
\
\ <br>

|                                                                                                                                                                                                                                                                                                                                                                              |
| :--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------: |
| *For any feedback or questions regarding this article (Illumina Knowledge Article #1538), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000001538%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/rna-library-prep/library-preparation-rna-library-prep-reference_material-list/000001538.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.