0.8X bead cleanup to remove adapter dimers from Illumina Single Cell 3’ RNA Prep libraries

Residual adapter dimers in final libraries can reduce sequencing efficiency and negatively impact data quality. The recommended thresholds are ≤0.5% for patterned flow cells and ≤5% for non-patterned flow cells.

For optimal performance, Illumina recommends sequencing Illumina Single Cell (ISC) libraries that are completely free of adapter dimers, as even small amounts can divert reads from desired library fragments.

The following protocol is adapted from the Clean Up Library step of the ISC 3’ RNA Prep workflow. Adjust volumes based on the initial and desired final library yield.

Pre-cleanup Considerations

• Use this protocol only if the library concentration is sufficient as additional magnetic bead purifications can further reduce yield. Confirm the minimum required yield for optimal sequencing performance by referencing the appropriate denature and dilute protocol and the Illumina Single Cell 3' RNA Preparation library sequencing recommendations.

• Review bead handling and magnetic cleanup best practices in the Training Packet

• This additional purification step is not validated by Illumina, and results may vary.

Customer-Provided Materials

• Magnetic side selection beads (eg, Illumina Purification Beads)

• Absolute ethanol, molecular biology grade (EtOH)

• Nuclease-free water (sterile)

• 10 mM Tris, pH 8

• 0.2 ml PCR 8-tube strips and caps

• 0.2 ml magnetic stand

• 20 μl and 200 μl sterile, filtered pipette tips (using low-retention tips can minimize loss)

Preparation

1. For each reaction, prepare 400 μl fresh 85% EtOH solution from absolute EtOH.

2. Thaw the Illumina Single Cell library stored at ‑25°C to ‑15°C fully on ice.

3. Measure the volume of the library with a 20 μl pipette, add nuclease-free water to achieve a 100 μl total reaction volume.

Workflow tip: After adding nuclease-free water, use a 200 μl pipette to verify that the final reaction volume is 100 μl. Make sure the entire volume is dispensed back into the PCR tube to prevent sample loss.

1. Cap and briefly centrifuge the sample tube.

0.8X Magnetic Bead Cleanup Instructions

1. Resuspend magnetic beads by vortexing.

2. For 100 μl reaction volumes, add 80 μl magnetic beads. If needed, adjust the magnetic bead volume for a 0.8X ratio of magnetic beads.

⚠️ Extremely low-volume cleanups are not recommended.

1. Pipette 15 times at the 170 μl stroke to mix.

⚠️ Inadequate mixing of the sample and magnetic beads can cause variability in size selection. Ensure thorough mixing for consistent results.

1. Incubate at room temperature for 5 minutes.

2. If any magnetic beads remain on the lid or side of the tube, cap tubes and pulse centrifuge briefly.

3. Place on the magnetic stand and wait until the liquid is clear (~5 minutes).

4. Without disturbing the beads, remove and discard all supernatant from each tube.

5. Wash beads as follows.

a. Add 200 μl fresh 85% EtOH.

b. Wait 30 seconds.

c. Without disturbing the pellet, remove and discard EtOH.

1. Wash the beads a second time.

2. Without disturbing the beads, use a 20 μl pipette to remove residual EtOH.

3. Air-dry until a slight gloss remains on the bead surface (typically ~2 minutes in strip tubes). Depending on humidity level, air-drying can take up to 5 minutes.

⚠️ Overdrying the beads can result in cracks and decrease elution efficiency.

Workflow tip: Immediately resuspend the magnetic bead pellet as soon as it only has a slight gloss remaining. When the magnetic bead pellet no longer looks shiny, it can quickly become cracked.

1. Remove from the magnetic stand.

2. Add 21 μl 10 mM Tris, pH 8 (or nuclease-free water).

⚠️ Elution volume should be sufficient to allow the beads to properly bind to the magnet.

1. Pipette 10 times at the 20 μl stroke to resuspend.

2. Incubate at room temperature for 5 minutes.

Workflow Tip: If beads are accidentally overdried and showing cracks while resuspending, gently pipette up and down periodically (~ every 30 seconds) during this 5-minute incubation off of the magnetic rack.

1. Place on the magnetic stand and wait until the liquid is clear (~2 minutes).

2. Without disturbing the beads, transfer 20 μl supernatant to a new PCR tube strip.

⚠️ Store ISC 3’ RNA Prep libraries at -25°C to -15°C and sequence within 30 days of completing the original Clean Up Library step.

Additional Resources

• Perform quality checks on re-cleaned ISC 3' RNA Prep libraries as outlined in assessing library QC and library QC best practices.

• For future workflow guidance, review the Illumina Single Cell 3’ RNA Prep Training Packet.

• For troubleshooting support, contact Illumina Technical Support.

For any feedback or questions regarding this article (Illumina Knowledge Article #9534), contact Illumina Technical Support [e-mail].