> 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/general/library-preparation-general-reference_material-list/000001538.md).

# 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):\
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. Mix thoroughly as follows. **It is critical to mix thoroughly**.

* \[HS] Shake at 1600 rpm for 20 seconds.
* \[LS] Pipette up and down.

**Remove rRNA procedure:**

1. Vortex RRB until well-dispersed. **The beads must be completely resuspended before addition to the plate**.
2. Add 35 µl RRB to each well of the RRP plate.\
   **Note**:In this step, the beads are added to a new plate, and the sample are added to this new plate on top of the beads. 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. **This procedure is required**.
3. Transfer all (20 µl) from each well of the BRP plate to the corresponding well of the RRP plate, and then mix thoroughly as follows.

* \[HS] Shake at 1000 rpm for 1 minute.
* \[LS] Pipette up and down.
  * **Notes for pipette mixing**:\
    \- 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.\
    \- 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 critical points**:

* Accidentally taking some rRNA Removal Beads (RRB) beads into the RNAClean bead purification step will add rRNA back into the samples. The indicated transfers may not be sufficient if care is not taken to completely remove the beads.
* **Never freeze rRNA Removal Beads**.
* **It is important to follow the protocol (including all notes) step-by-step**.

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| *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)*.* |


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