Library Preparation > Illumina Single Cell RNA Prep > Reference Material

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

• Assessing cDNA QC in Illumina Single Cell 3’ RNA Prep

• Assessing library QC in Illumina Single Cell 3’ RNA Prep

• Best Practices for Nuclei Isolation with alternative nuclei isolation protocols for Illumina Single Cell

• Best practices for cell preparation for Illumina Single Cell prep

• Best practices for using Illumina Single Cell Nuclei Isolation Kits for Illumina Single Cell kits

• Cell input and volume loading recommendations for Illumina Single Cell 3’ RNA Prep

• Differences between V4.0PLUS PIPseq and Illumina Single Cell (formerly PIPseq V)

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

• Illumina Single Cell 3' RNA Preparation library sequencing recommendations and guidelines

• Representative cDNA QC traces for Illumina Single Cell 3’ RNA Prep

• Sample Enrichment Best Practices for Illumina Single Cell 3' RNA Prep

• The importance of pilot studies for Illumina Single Cell RNA Preparation prior to workflow changes

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.

Ensuring that cDNA meets quality expectations during the Check QC Product step is critical for obtaining optimal library QC and sequencing results in Illumina Single Cell 3’ RNA Prep.

Refer to Illumina Single Cell 3’ RNA Prep cDNA QC best practices to confirm accurate QC assessment.

cDNA QC Yield Expectations

• cDNA yield depends on several factors, including sample type, cell/nuclei loading, and PCR cycle number used during the Assess cDNA Quality step.

• Adjust the QC Amplification program to optimize for different input quality and concentration.

• If cDNA QC yields ≥ 200 ng/µl in T10, T20, or T100 kits (not applicable to T2 kits), reduce the number of PCR cycles in the Amplify Library step by 1-2.

• For more information on recommended PCR cycling parameters, refer to High and Low RNA Sample types and choosing the number of cDNA QC and Amplify Library cycle numbers in Illumina Single Cell RNA prep kits.

cDNA Fragment Analysis Expectations

The cDNA average fragment size should be > 500 bp when the fragment analyzer region table is set from 200 bp to 5000 bp to proceed with Library Prep.

cDNA peaks, distribution, and average fragment size vary based on sample type and experimental condition(s). For example traces from various sample types, refer to Representative cDNA QC traces for Illumina Single Cell 3’ RNA Prep.

Figure 1. Representative human/mouse cell mixture (HEK 293T/NIH 3T3) using a High Sensitivity D5000 ScreenTape.

If the average cDNA average fragment size is <500 bp, it may indicate significant cDNA degradation.

Figure 2. Degraded cDNA QC product from mouse pancreas nuclei that does not pass QC specifications (<500 bp).

Refer to Section 3.2 Quality Checks and Section 3.3 cDNA Degradation in the Illumina Single Cell 3’ RNA Prep Training Packet on the Illumina Single Cell Prep Support Page for additional guidance on assessing degraded cDNA.

Library Yield Expectations

Library yield depends on several factors, including sample type, cell/nuclei input, and PCR cycle number used during the Amplify Library step of the Illumina Single Cell (SC) 3' RNA Prep workflow.

Refer to Illumina Single Cell 3’ RNA Prep library QC best practices to confirm accurate QC assessment.

Adjust the number of PCR cycles used during the Amplify Library program to optimize for different input quality and concentration. Review the Illumina Single Cell 3' RNA Preparation library sequencing recommendations and confirm the minimum yield required for optimal sequencing performance.

For more information on recommended PCR cycling parameters, refer to High and Low RNA Sample types and choosing the number of cDNA QC and Amplify Library cycle numbers in Illumina Single Cell RNA prep kits.

Final Library QC Expectations and Assessment Best Practices

The final Illumina SC 3' RNA Prep library is expected to have an average fragment size ranging between 370-550 bp when the region table is set from 200 to 800 bp.

Library distributions and average fragment size may vary based on sample type and experimental conditions. Refer to Representative library QC traces for Illumina Single Cell 3’ RNA Prep for additional examples from different sample types.

Figure 1. Representative fragment analysis of an Illumina Single Cell 3’ RNA Prep T2 library from a 5,000-cell HEK 293T/NIH 3T3 mixture using a HS D1000 ScreenTape.

If there are large adapter peaks remaining in the final library, these can significantly reduce sequencing efficiency. If these are present, Illumina recommends performing an additional 0.8X magnetic bead cleanup. This extra size selection is not recommended if the library prep concentrations are low. Contact Illumina Technical Support for additional information.

Refer to Section 3.2: Quality Checks of the Illumina Single Cell 3’ RNA Prep Training Packet on the Illumina Single Cell Prep Support page for information on final library assessment. Always check the Illumina support site for the latest version.

It is not required to use the Illumina Single Cell Nuclei Isolation kit for sample preps of nuclei to be compatible with the Illumina Single Cell workflow. Alternative workflows from other vendors or lab developed workflows can be used as long as the recommendations in the Training Packet for Alternative Nuclei Isolation protocols are followed. Recommendations are reviewed below.

Recommendations

• When using alternative nuclei isolation protocols, it is necessary to include final concentrations of 0.8 U/μl RNase inhibitor and 1X protease inhibitor into the nuclei extraction buffer/nuclei lysis buffer during tissue lysis. Failure to do so can result in degraded cDNA.

• After nuclei extraction is complete, centrifuge the nuclei suspension (preferably, 500 × g for 5 minutes at 4°C) to pellet and aspirate the supernatant. Use a P200 to carefully remove as much supernatant as possible without disturbing the pellet.

Important note:using a fixed angle centrifuge for pelleting nuclei for wash steps can result in excessive sample loss. Tubes compatible with swinging bucket centrifuges should be used to result in a flat pellet.

• Add 1 ml of 1X Nuclei Suspension Buffer (1X NSB), mix well and spin at 500 × g for 5 minutes at 4°C.

• Next, aspirate supernatant without disturbing the pellet and resuspend nuclei using 1X NSB.

Note: centrifuge speeds/times listed may need to be adjusted for non-mammalian sample types.

It is possible to use other kits or alternative protocols for nuclei isolation, but the extra time-consuming steps to minimize large debris to prevent clogging of microfluidic devices (eg, sucrose or other density gradients, magnetic bead purifications) can be harsh on the nuclei. This can increase nuclei leakage and prevent transcripts from paranuclear membranes from being captured.

Using filter steps is a gentle method for debris removal and is optimal for PIPseq technology, which has no instrument clogging issues from cellular or nuclear debris. After Dounce homogenizer steps, it is recommended to use two filter steps to remove debris. It is recommended to start with a 40 μm gravity filter. If the first filter step takes more than ~3 minutes, optionally use an Uberstrainer with negative pressure for the first filter step (but this will let through more debris), then immediately follow with a 40 μm gravity filter step to remove any remaining debris, which should then go much quicker.

For the second filter step, it is recommended to use a smaller sized filter, such as a 10 μm Uberstrainer while applying negative pressure. For nuclei from mouse brain or other larger nuclei sample types, a 20 μm Uberstrainer may be used. The faster this step can be completed, the better quality the isolated nuclei will be. Using negative pressure is preferable to gravity filtration.

When using an alternative nuclei isolation kits or protocols, the RNase inhibitor is user supplied when adding it to the nuclei extraction buffer or when formulating the 1X Nuclei Suspension Buffer (NSB). The Illumina Single Cell 3’ RNA kits provide enough RNase inhibitor to add with the samples into the PIP tube during Capture and Lysis.

Recommended alternative RNase Inhibitors include: Protector RNase Inhibitor (40 U/μl) and RiboGrip RNase Inhibitor (220 U/μl) are optimal for nuclei, with RiboGrip being the best choice for challenging sample types, such as those with lower RNA content and/or higher endogenous RNase (eg plants).

Enough Bovine Serum Albumin (BSA) is provided in the kits for the final nuclei suspension, but if users would like to include BSA in the 1X NSB used for wash steps (this is optional, and not used internally), additional BSA (molecular-biology grade) should be purchased.

DSP-Methanol fixation is an alternative option that can be used to help protect the mRNA in isolated nuclei before the Illumina Single Cell workflow and is especially recommended if it is not possible to proceed immediately to Capture and Lysis following nuclei isolation.

Refer to the Sample Preparation section of the Training packet for more information.

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

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.

The Illumina Single Cell Nuclei Isolation kit provides a pre-formulated set of reagents for isolation of nuclei from frozen tissue and is designed to be compatible with various tissue types. The isolated nuclei are compatible with Illumina Single Cell 3’ RNA Kits.

The Illumina Single Cell Nuclei Isolation Kit (formerly known as the Fluent V Nuclei Isolation kit) has been validated for frozen mammalian tissue.

Certain reagents and steps may be too harsh for use with other sample types. It is not recommended for use with non-mammalian sample types. The lysis incubation time, the number of steps using a Dounce homogenizer, and the centrifuge speed/time used to pellet nuclei might need to be adjusted for fresh mammalian tissue and performance is not guaranteed. Contact Illumina Technical support at techsupport@illumina.com for further recommendations if interested in isolating nuclei from cell lines or cells in culture.

Assessing Nuclei Quality

The quality of isolated nuclei significantly impacts downstream single-cell workflows. Nuclei quality should always be assessed before input into Illumina Single Cell and the nuclei isolation workflow should be repeated if sufficient quality is not obtained.

Visual inspection of nuclei is recommended to determine the quality and the concentration of a nuclei suspension before input into Illumina Single Cell 3’ RNA kits. Quantify generated nuclei suspensions with a fluorescent nucleic acid stain, like Acridine Orange or Propidium Iodide (AO/PI), with a fluorescence-capable automated counter. Always conduct replicate counts to verify accuracy. Use of trypan blue is not recommended.

A high-quality nuclei suspension will have minimal debris and aggregates (Figure 1A). For tissues with high amounts of debris, a smaller tissue input is recommended to reduce debris carryover. High-quality nuclei with intact membranes will appear round and smooth (Fig 1 B) while nuclei with compromised membranes will appear disjointed with an indication of blebbing (Fig 1 A).

General Recommendations for Nuclei

The quicker cells are processed to encapsulated nuclei, the better. Minimal centrifugation and keeping the preps ice cold the entire time is recommended. (Keep all buffers, reagents, equipment, consumables, and plasticware on ice throughout the isolation.) When isolating nuclei for many samples, it is best to prep samples in small batches through Capture and Lysis (10-15 minutes of hands-on time), then use the stopping point to prep another batch of samples. The samples can then be processed together through the remaining Illumina Single Cell workflow steps to eliminate batch effects. Try to minimize the amount of time between completing nuclei isolation and starting Capture and Lysis. After isolation, nuclei will start to degrade and clump.

Do not freeze and store isolated nuclei (freeze/thaw cycles disrupt the nuclear membrane) unless fixing nuclei first according to the demonstrated DSP-Methanol Fixation Protocol for Nuclei for Illumina Single Cell. Refer to the Fixation section of the Training Packet for further information.

Always conduct replicate counts to verify accuracy. It is not recommended to use trypan blue, as debris can easily be counted as nuclei, which will overestimate the count (leading to a lower-than-expected capture rate), especially when size gating is not implemented.

Size Gating Recommendations (Nuclei)

Size gating recommendations using an automated fluorescent cell counter with nuclei isolated from various mouse tissue types:

Minimum size gate: 4 µm

Maximum size gate: 11 µm (16 µm for brain)

For more information refer to the Training Packet.

Cell and Nuclei Loading Parameters

Cell and nuclei loading in Illumina Single Cell 3’ RNA Prep kits is optimized to balance high recovery rates and data quality. The table below summarizes optimal loading parameters by kit size.

*Exceeding the recommended input increases the risk of a multiplet rate above 8%.

Notes:

• Always include RNase inhibitor in the total loading volume.

• Refer to reference guides for loading volumes to ensure optimal capture efficiency.

Cell and Nuclei Loading Volume Considerations

• Adjust the target concentration if increasing or decreasing input.

• The total loading volume, including RNase inhibitor, must not exceed the specified limit for each kit size. Exceeding recommended volumes may reduce capture efficiency.

• If cells or nuclei cannot be concentrated to the target level, a lower concentration is generally acceptable as long as the total volume remains within recommended limits.

The high level advantages of Illumina Single Cell (Formerly PIPseq V) when compared to PIPseq V4.0PLUS are covered in this article while the full details are covered in the BioRxiv pre-print in the results section.

High-level benefits:

• New sequencing-artifact reducing PIP design for more usable data.

• New optimized chemistry includes novel reverse transcriptase that improves reverse transcription and template-switching efficiencies.

• Increased sensitivity from using the full cDNA yield for library preparation after limited-cycle amplification of the cDNA (5 cycles only).

• The random enzymatic fragmentation during library preparation results in Intrinsic Molecular Identifiers rather than UMIs to remove duplicates.

• 10 bp indexes that are pre-combined in a strip frame for multi-channel pipetting ease.

• The Clean Up Library steps use a single-sided size selection (no double-sided size selection required).

• The PIPseq platefuge microcentrifuge requirement for processing T2 reactions has been eliminated, but labs may continue to use it if preferred.

Protocol Changes:

• Changes to master mix volumes for cDNA Synthesis and Amplify cDNA.

• Assess cDNA Quality steps are completed after Clean Up cDNA using leftover supernatant. Leave the tubes on the magnetic rack and do not discard stock reagents used for Amplify cDNA, as they will be used again for the QC.

• Library Preparation steps must be started within 72 hours of Clean up cDNA. Longer storage of limited-cycle cDNA can result in cDNA degradation.

• There are changes to the Fragmentation, End Repair and A-Tailing, and the Ligation thermal cycler profiles; be sure to update these if the v4.0PLUS programs are saved.

• The WTA primer used for Amplify cDNA must be diluted at a 1:10 ratio before adding it to the master mix for T2 kits only. It should not be diluted during Assess cDNA Quality.

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 high amplicon-contaminated environments.

⚠️ Even if cDNA QC results from the Check QC Product step are not available, proceed with Library Prep 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 and Assessing cDNA QC in Illumina Single Cell 3’ RNA Prep for more details on proper cDNA QC product assessment.

Resolution:

If library preparation fails despite obtaining QC-passed cDNA, the 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 methods avoids having to restart from cell loading with a new sample.

Contact Illumina Technical Support for more information.

Illumina Single Cell 3’ RNA preparation libraries are composed of standard Illumina paired-end constructs that begin with P5 and end with P7.

Read 1 contains barcode information and must be sequenced > 45 bases.

Read 2 contains gene expression information and must be sequenced > 72 bases.

The libraries are dual-indexed with 10-base i5 and i7 indexes.

Together this combines for a total of a minimum of 137 cycles needed to sequence Illumina Single Cell 3’ libraries.

Read Depth Recommendations

The recommended sequencing read depth for the Illumina Single Cell 3’ RNA Kit sizes of T2, T10, and T20 is 20,000 reads per input cell. The required read depth for kits loaded at the recommended cell/nuclei loading is as follows (M = million):

• For T2, 5,000 cells loaded requires 100 M reads per T2 sample

• For T10, 17,000 cells loaded requires 340 M reads per T10 sample

• For T20, 40,000 cells loaded requires 800 M reads per T20 sample

The recommended sequencing read depth for the T100 kit is 10,000 reads per input cell.

• For T100, 200,000 cells loaded requires 2 billion reads per T100 sample

After the users first run with their specific sample type and a specific kit size, users can evaluate capture rate and sequencing saturation metrics to determine if sequencing depth can be adjusted in future experiments based on sample type and experimental needs.

Final Loading Recommendations

The final library loading concentrations listed are general recommendations for sequencing Illumina Single Cell 3’ RNA Prep libraries. The following concentrations may need to be adjusted to optimize performance:

• NextSeq 500/550 recommended final library loading concentration 1.6 pM including ≥ 1% PhiX.

• NextSeq 2000 recommended final library loading concentration: 550 pM including ≥ 1% PhiX.

• NovaSeq 6000 final library loading concentration 210 pM including ≥ 1% PhiX (equivalent to Pooled Loading Concentration of 1.05 nM).

• NovaSeq X Series final library loading concentration 190 - 200 pM including ≥ 2% PhiX.

  

It is recommended to pool libraries together from all experimental conditions before single-cell sequencing with an Illumina sequencing system, as this will minimize batch effects and can help with index color balancing.

Refer to the Supplemental Enrichment and Amplification (SEA) kit user guides for pooling recommendations for the applications that generate a targeted library to be sequenced with Illumina Single Cell 3’ RNA libraries.

A minimum of 1% PhiX is required in the final library loading pool. If a NovaSeq X Series instrument is being used, a minimum of 2% PhiX should be used.

For more information, refer to the Sequencing section of the Illumina Single Cell Training Packet.

cDNA peaks, distribution, and average fragment size can vary based on sample type and experimental condition(s).

The following TapeStation-generated traces serve as representative examples of satisfactory cDNA QC profiles generated during the Check QC Product step in the Illumina Single Cell 3’ RNA Prep workflow.

Human peripheral blood mononuclear cells (PBMCs)

Human HEK 293T & mouse NIH 3T3 cell line mixture

Mouse frozen tissue: unfixed brain nuclei

Mouse frozen tissue: DSP-methanol fixed brain nuclei

Mouse frozen tissue: unfixed mouse diaphragm

Mouse frozen tissue: unfixed mouse heart

Mouse frozen tissue: unfixed lung nuclei

Mouse frozen tissue: DSP-methanol fixed lung nuclei (1-month storage)

For example final library traces produced using similar sample types, see Representative library QC traces for Illumina Single Cell 3’ RNA Prep

For more information related to cDNA QC product assessment refer to:

• Illumina Single Cell 3’ RNA Prep cDNA QC best practices

• Assessing cDNA QC in Illumina Single Cell 3’ RNA Prep

• Illumina Single Cell 3' RNA Prep Training Packet

Fluorescence-activated cell or nuclei sorting (FACS or FANS) and magnetic-activated cell sorting (MACS) can be beneficial for the enrichment of target cell populations and can facilitate the exclusion of dead or damaged cells. Enrichment steps also add significant time and can stress the cells, causing an overall decrease in cell health. It can be necessary to perform a second live/dead cell sort following cell sorting to ensure sufficient cell viability.

Dead Cell Removal Kits

It is not recommended to move forward with cells that are less than 75% viable. Adding additional cell wash steps is best if only minor viability improvement is needed. If significant improvement is required, it is recommended to either optimize sample preparation processes and reduce sample preparation time, or to incorporate a Dead Cell Removal Kit (eg, Akadeum Life Sciences, Miltenyi Biotec). These kits require starting with a high number of cells (500,000 minimum cells for Akadeum and 1 million cells for Miltenyi).

FACS and FANS

It is common for Fetal Bovine Serum (FBS) to be used in pre-sorting buffers and in collection buffers. As an alternative to FBS (which is inhibitory), Bovine Serum Albumin (BSA) can be used in buffers and for tube blocking. It is critical to complete the cell or nuclei wash step according to the user guide after sorting if the sorted samples contain any potentially inhibitory reagents such as FBS.

If working with low cell or nuclei numbers and there will be no inhibitory reagents in the final suspension, a low volume of Cell Suspension Buffer or 1X Nuclei Suspension Buffer may be used in the collection tube (200-400 µl). Be sure to include the recommended final concentrations of RNase inhibitor and BSA for the sample type being worked with as described in this section. The 1-2 ml wash step may be eliminated if there are no potential inhibitors. Do a post-enrichment count and determine the count for the entire tube. The cells or nuclei may then be concentrated by centrifugation to pellet the samples, then removing a safe volume of the supernatant so as not to disturb the cell or nuclei pellet. Make sure there is sufficient volume of supernatant remaining in the tube to load into the PIP tube for the kit size being used. Resuspend the samples and determine the new count by adjusting the tube count by the new final volume.

Illumina Single Cell 3' RNA Prep technology is optimized for efficient and sensitive transcript capture from live cells, and therefore, quick isolation and mild dissociation of cell types is essential and should be confirmed in a pilot experiment before committing to the actual experiment. It is also helpful to complete a pilot prior to scaling up, increasing sample number, or using Supplemental Enrichment and Amplification (SEA) kit applications. Completion of a pilot study before scaling up is especially important when working with any new sample types. Pilot studies are also valuable for labs new to single cell sequencing.

General Pilot Study Recommendations

The T2 kits have the same chemistry as the higher cell capture kits, so T2 kits can be used to assess each step of the scRNAseq process and complete any optimization needed, before scaling up to larger reactions that may require a significantly higher read depth and higher output sequencing runs. The following list of recommendations can help with preparation for an effective pilot study before scaling up, when working with a new sample type, or for users new to single cell.

• Purchasing a T2 kit along with the T10, T20, or T100 kits is an economical way to complete a pilot study.

• Processing a small number of samples (1-4 samples) as opposed to starting with large studies with many treatments or conditions.

• Optimizing steps of cell or nuclei preparation for a new sample type including:

  • Dissociation

  • Accurate quantitation of cell/nuclei counts

  • Cell viability assessment in Illumina Single Cell - Cell Suspension Buffer

  • Proper pipetting force

  • Centrifugation speeds

  • Labeling cells

  • Enrichment

  • Use of third party kits to remove debris and/or dead cells

• Comparing wild type (WT) versus treated or mutant conditions

  • Treated/mutant groups may have lower quality mRNA that is inherent to their biology.

  • Discovering this during a small pilot prior to a large-scale study allows for a more informed experiment.

  • It may be necessary to prepare 2+ Illumina Single Cell reactions for the treated/mutant group to achieve the same number of captured cells as the WT.

  • Technical replicates of treated/mutant groups should be sequenced in the same sequencing run and merged using analysis software to be analyzed as a single sample.

• Completing Illumina Single Cell T2 reactions through cDNA QC and Library QC successfully without sending for sequencing can be economical.

  • If taken through sequencing, this will provide even more information to help guide the larger experiment design when scaling up.

  • Review key sequencing metrics to verify data quality is good before deeper sequencing with larger cell numbers including: - Captured cell count - % reads mapped - % reads in cells - % mitochondrial reads - Duplication rate - Median Genes/cell - Median Transcripts/cell

• Alternatively, after completing all sample preparation processes upstream of the Illumina Single Cell workflow, the total RNA can be isolated and run with an Agilent TapeStation RNA Assay to evaluate the quality and to verify the total RNA is not degraded (RIN value ≥ 7 is recommended).