SureCell WTA 3´ Library Prep Kit FAQs

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  • Input

  • 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.

  • Protocol

  • 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.
  • Library Evaluation

  • 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.