SureCell WTA 3´ Library Prep Kit FAQs

The workflow prepares 3’-tagged RNA-Seq libraries from single cells for whole transcriptome gene profiling analysis on Illumina sequencing systems. The protocol requires a Bio-Rad ddSEQ Single-Cell Isolator (SCI) and reagents provided in the Illumina | Bio-Rad SureCell WTA 3' Library Preparation Kit for the ddSEQ System to isolate single cells and barcode individual transcriptomes.

The SureCell WTA 3' Library Preparation Kit can only be used with the Bio-Rad ddSEQ Single-Cell Isolator. Before proceeding, confirm that the ddSEQ Single-Cell Isolator is installed and operating properly. Refer to the Bio-Rad ddSEQ Single-Cell Isolator Instruction Manual to learn how to operate your ddSEQ Single-Cell Isolator instrument, best practices for use, and maintenance.  

Each ddSEQ Single-Cell Isolator cartridge has 4 cell input wells that can be loaded with up to 4 unique samples, for an average cell output of 300 single cells per sample.

No, the ddSEQ Single-Cell Isolator coencapsulates single cells and barcodes into subnanoliter droplets to create a highly parallelized library prep for single-cell analysis.

Create a single-cell suspension during cell preparation for loading on the ddSEQ Single-Cell Isolator. Make sure that cells are well suspended and viable before loading on the cartridge.

SureCell WTA 3’ 8 sample and 24 sample kits are kitted for different sample numbers. 

The 8 sample configuration contains 4 boxes.

The 24 sample configuration contains 10 boxes.

Yes, the protocol generates stranded libraries. Strand specificity is achieved by replacing dTTP with dUTP in the Second Strand Buffer (SSB). The incorporation of dUTP in second strand synthesis effectively quenches the second strand during amplification, since the polymerase used in the assay will not incorporate past this nucleotide.

One year from the date of manufacture. The kit label provides an exact expiration date. Illumina guarantees at least three months from the date of receipt.

Rainin pipettes and pipette tips are required for the following steps in the SureCell workflow:

• Prepare Single-Cell Suspension (P200 or P1000 Single Channel)
• Assess Cell Count and Viability (P20 Single Channel)
• Prepare Cell and Barcode Suspension Mixes (P20 Single Channel, P200 Single Channel)
• Isolate Single Cells (P20 Single and Multichannel, P50 Multichannel, P200 Multichannel)

See the Consumables and Equipment section in the SureCell WTA 3’ Library Prep Reference Guide for more information.

Cleanup procedures have only been optimized and validated using the magnetic stands specified in the SureCell WTA 3’ Library Prep Reference Guide. Two different types of magnetic stands are required and both are needed to complete the protocol.  These magnets are not interchangeable and comparable performance is not guaranteed when using other magnets. If you choose to use a different magnet, you may need to optimize incubation times and take care to qualify the length and yields of your libraries to ensure successful results.

Two major aspects can make a magnet suboptimal for this protocol:

1. If the magnet is too weak and doesn’t sufficiently pull beads out of solution
2. If the geometry of the magnet makes it difficult to remove the entire supernatant cleanly. Removing beads with supernatant during clean up steps can drastically reduce final library yields and lead to poor sequencing results.

The SureCell WTA 3’ Library Prep Reference Guide includes a Verified Protocol for running mixed species control samples in a 1:1 mix to assess HEK and 3T3 mixed-species crosstalk and doublet cells. This protocol can be used as a control in parallel with other samples.

Illumina and Bio-Rad have validated the Bio-Rad C1000 and Bio-Rad T100 thermal cyclers. Other thermal cyclers may require additional optimization.

The workflow has been tested with commercially available cell lines, Mouse (A20, NIH-3T3) and Human (HEK-293 , BJ), Jurkat, K562, and primary keratinocyte for a variety of cell diameters and IFC size classes. The protocols provided may be used as a starting point for many adherent or suspension cell lines. However, the dissociation, straining, washing, quantitation, and dispensing buffer conditions may require additional optimization and specific techniques appropriate for your cell type or cell source.

When performing the SureCell WTA 3' Library Prep Kit assay for the first time or when working with a new cell type, we recommend preparing additional cells that can be used to optimize cell dissociation conditions before starting a full experiment. Consider these methods when optimizing cell dissociation:

• Types of trypsin or alternative enzymes typical for your cell type
• Incubation time and temperature for trypsinization
• Intensity of pipetting
• Types (diameter) of serological pipettes or micropipette tips to be used

A verified protocol for a Mixed Species Control experiment can be found in the SureCell WTA 3’ Library Prep Reference Guide, describing a control protocol that can be used to assess your workflow.

Load a single-cell suspension with concentration of 2500 cells/µl ± 10% variance (2250–2750 cells/μl).

High viability (>95%) and cell integrity is necessary. Dead or damaged cells can release nucleic acids into the cell suspension buffer. This background signal from these cells remains through subsequent steps, and may impact the quality of the resulting analysis. Trypan Blue is a user-supplied consumable that is needed to assess viability.

Yes, single-cell encapsulation by the ddSEQ Single-Cell Isolator requires complete dissociation of cells to form a single-cell suspension prior to droplet generation. Cell aggregates present in the suspension will significantly increase the probability of cell doublets or multiplets, making data interpretation potentially more difficult. Depending on the method or cell counter device used, multiplets also can affect the accuracy of cell counting.

Accurate cell count is critical to achieve target cell throughput and to avoid cell multiplets. Illumina and Bio-Rad have validated and recommend the use of an automated cell counter (Bio-Rad TC20) for accurate cell counting. Comparable performance is not guaranteed when using other cell counters.

Cells must be kept cold at all times to prevent cell clumping before droplet formation. Follow protocol instructions carefully for cell and reagent handling to determine if a step is performed at room temperature or on ice.

We highly recommend that the single-cell suspension be prepared as close as possible to loading the ddSEQ Single-Cell Isolator. If you require staging, place the suspended cells on ice for up to 1 hour before loading, but make sure the cells are in a single cell suspension before loading.

One ddSEQ cartridge can be loaded with up to 4 unique samples. If you want to only load 1 sample, fill the remaining input wells with the Bead Suspension Mix and the Cell Suspension Mix. 

After cells have been prepared, there are no safe stopping points until Second Strand Synthesis has begun. Proceed immediately to each step in the protocol. Delays during cell preparation and handling can lead to sample failure. Make sure that you have all the required consumables and equipment before you begin.

After Second Strand Synthesis, there are Safe Stopping Points after Synthesize Second Strand cDNA, Clean Up cDNA, Amplify Tagmented cDNA, and Clean Up Libraries.

The ddSEQ Single Cell Isolator can prepare 1 ddSEQ cartridge at a time. However, you can process a second cartridge after you have completed the “Isolate Single Cells” step for the first cartridge.  Refer to the SureCell WTA 3’ Library Prep Reference Guide for processing 2 cartridges.  Contact Illumina Technical Support for processing more than 2 cartridges.

The reagents in this protocol have color-coded caps to identify the associated suspension mixes with the colored wells in the ddSEQ cartridge holder for ease of loading.

• Red caps identify reagents used to create Cell Enzyme Mix
• Blue caps identify reagents used to create Barcode Suspension Mix

No, AMPure XP beads are not used in this preparation. Instead, Purification Beads (SPB) are supplied with the kit for clean-up steps.

Plastic seals can generate static and impact encapsulated samples, which may increase chance of multiplets. 

Yes, the indexed DNA Adapters (N7XX) are required for addition of the full length adapter sequence. Without this sequence, the library will not be compatible with Illumina sequencers.

Use a different DNA adapter for each well regardless if the cell samples are the same or different. The SureCell WTA 3’ Library Prep Kit provides enough indices to use a different index for each well, which allows tracking of wells through analysis.

The protocol uses Nextera indexes (N7XX).  The index names can be found in the SureCell WTA 3’ Library Prep Reference Guide. Index sequences are listed in the Illumina Adapter Sequences Document.

The ddSEQ takes approximately 5 minutes to generate droplets. The instrument indicator light flashes green to indicate cell isolation is in progress. Isolation is complete when all 3 indicator lights are solid green.

Upon opening the instrument, encapsulated samples will appear cloudy in the output wells of the ddSEQ cartridge. Note if any wells look clear or empty, as droplet generation may have failed at those positions.

Before starting this assay, watch the SureCell WTA 3' Library Prep: Best Practices training video on the Training page. This video covers key steps and best practices for the SureCell WTA 3’ Library Prep Kit workflow.

Also review the Bio-Rad ddSEQ Single-Cell Isolator Instruction Manual for Best Practices for Loading the Cartridge and Droplet Transfer.

Some key steps are:

1. When removing encapsulated samples from cartridge output wells, gently and slowly (5 sec/50 µl) pipette samples.
2. In the “Break Emulsion” step, visually examine samples to see that 2 layers have formed (an oil layer on bottom and an aqueous layer on top). Note if any wells have only 1 layer. Reagents in the next steps will be added or mixed only in the top, aqueous layer.
3. In the “Clean Up First Strand Synthesis” step, Purification Beads (SPB) will be added and mixed in the aqueous layer. Make sure to mix the beads with the aqueous layer robustly, so that the aqueous layer is homogenously brown on top. If you see a gradient in the aqueous layer, continue mixing before proceeding to the next step.

Yes, there are multiple points where you can check your progress through the workflow:

• After the Isolate Single Cells step on the ddSEQ, visually confirm that samples in output wells have a cloudy appearance and note any wells that are empty or clear.
• After Clean Up cDNA, we recommend that you run 1 µl of undiluted library on an Agilent Technology 2100 BioAnalyzer using a High Sensitivity DNA chip. We have provided an example trace for comparison. Typical cDNA profiles range between ~400–8000 bp. cDNA yields at > 2 ng are sufficient to proceed to the tagmentation step.
• In the Assess Libraries step, run 1 µl of undiluted library on an Agilent Technology 2100 Bioanalyzer using a High Sensitivity DNA chip to determine the size and concentration of the final libraries. An example trace is provided for comparison. Typical libraries show a broad size distribution ~300–1000 bp. A wide variety of libraries can be sequenced with average fragment sizes as small as 450 bp or as large as 1200 bp. If you see small products present ranging from 100-300bp in size, do not proceed with sequencing. An additional bead clean up is highly recommended to remove these smaller products.

Typical library yield is 2-10 nM. Depending on your actual library yield, normalize samples to 2 nM.

The BioAnalyzer is recommended for quantifying cDNA after cDNA Clean Up and the final libraries before sequencing. The region lines should be set from 200-800 bp to determine average fragment size and library yield.  Illumina does not recommend using fluorometric assays like Qubit or qPCR for quantification of the final libraries.

Modified loading concentrations are provided in the SureCell WTA 3’ Library Prep Reference Guide and are based on BioAnalyzer quantification.  If you are quantifying with another method, you may need to optimize the loading concentration.

This library is compatible with the MiniSeq, MiSeq, NextSeq 500, HiSeq 2500, and HiSeq 3000/4000. Data requirements make the assay best suited for the NextSeq 500 and HiSeq platforms.

A paired end run must be set-up because Read 1 will sequence the cell barcode and UMI transcript sequence. Read 2 will sequence the cDNA insert. The recommended sequencing length is 68 x 75. Illumina recommends a 68 cycle length for Read 1 to avoid sequencing the Poly A tail.

The libraries are single indexed.

Yes, the SureCell WTA 3’ Library Prep Kit comes with the SureCell Sequencing Primer at 50 µM (in Box 3 of 4) that is compatible with only SureCell libraries and PhiX. The SureCell Sequencing Primer is used for Read 1 and is added to the custom primer position (consult the custom primer documentation for your sequencing instrument). If the SureCell Sequencing Primer is not used, the sequencing run will not be successful. See the SureCell WTA 3’ Library Prep Reference Guide for instructions on how to use the Sequencing Primer.

You will need to specify the use of the custom primer wells when setting up the sample sheet or setting up PrepTab in the BaseSpace Sequence Hub.

We recommend spiking in 1% PhiX control as a positive control for alignment and error rate calculations. For more information, see the PhiX Control v3 support page on the Illumina website.

Datasets are available on the BaseSpace Public Data page.

In BaseSpace Sequence Hub, use the SureCell RNA Single-Cell App to analyze your data.

For analysis, the Single-Cell RNA app supports multiple species, including human (hg19), mouse (mm10), rat (rn5), zebrafish (danRer7), fly (dm3) and c. elegans (de10). Human (hg19) and mouse (mm10) mixed species experiments are also supported.

Yes. Contact Illumina Technical Support for more information.

Use Illumina Experiment Manager (IEM) v1.13 or later. Sample sheets generated using IEM will contain the correct UMI settings for BaseSpace or bcl2fastq2 v2.18 or later.

The app is designed specifically of analysis of samples prepared with the SureCell WTA 3’ library prep kit. The app has not been validated for analysis of samples prepared with any other kit.

The SureCell RNA Single-Cell app is designed to analyze samples prepared using the SureCell WTA 3’ Library Preparation kit. This app performs read alignment, cell and transcript assignment, cell and gene counting, filtering, and calculates and reports single-cell metrics. The SureCell RNA Single-Cell workflow consists of the following major steps. Consult the SureCell RNA Single-Cell app user guide for a detailed breakdown of the workflow:

1. Align read 2 against the full reference genome using STAR
2. Generate an annotated BAM file with each alignment tagged with both the cell barcode and the UMI
3. Count UMIs to generate gene expression counts
4. Filter cells based on the number of UMIs per cell barcode
5. Calculate alignment, cell and gene metrics
6. Generate aggregate and per-sample PDF reports

The SureCell RNA Single-Cell app uses the STAR aligner to align read 2 against the whole reference genome. Each aligned read in the BAM file is tagged with the cell barcode and UMI sequences from read 1.

Major output files include aligned reads in BAM format, gene counts per cell and per sample, and PDF reports containing single-cell RNA metrics.  Consult the SureCell RNA Single-Cell app user guide for more detailed information.