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Pushing the limits of sensitivity for single-cell applications Q&A
Over the last decade, the RNA-seq field has grown tremendously thanks to surges in new technologies enhancing capabilities and boosting performance. With this in mind, SPT Labtech and Takara Bio examined the advantages of the SMART-Seq Single Cell Kit (SSsc) and how the industry-leading sensitivity of SSsc remains unmatched even when scaled with the mosquito HV genomics liquid-handling system.
In our on-demand webinar, "Pushing the limits of sensitivity for single-cell applications," Nidhanjali Bansal, MS; Chris Waddling, PhD; and Kamila Koprowska, PhD discussed how best to use SSsc on the mosquito HV liquid handler.
Continue reading for insights from the Q&A session or register to watch the on-demand webinar.
Does the SSsc kit include UMIs?
The SSsc kit does not contain UMIs but the SSsc PLUS kit does contain UDIs to provide greater confidence in sequencing on patterned flow cells.
Have you miniaturized the library preparation portion of the PLUS kit?
The SSsc library preparation kit has been miniaturized to 1/2 volume. Please contact email@example.com for more information.
How many cells can be multiplexed with the SSsc kit?
With the SSsc PLUS kit, 96 samples can be multiplexed in a single sequencing run.
What is the minimum number of single cells required to achieve a reproducible result with the SSsc kit?
With as few as 3 single cells, we see high reproducibility between samples with control RNA and with single cells from cell lines. Overall, the number of cells required to achieve statistical confidence in your single-cell experiment will be vastly influenced by the heterogeneity of your input and the question you are asking. For example, a sample with a mix of cell types (i.e., PBMCs) or with a wide range of expression patterns (i.e., tumor cells) may require greater input numbers to achieve statistical confidence due to the noise in the sample. Because of the high reproducibility of the SSsc kit with uniform sample types, you can be confident that the variance in the data is true biological variance in your cells.
In a pseudo-bulk setup with 30–50 FACS-sorted cells in one well with lysis buffer, would you recommend using the SSsc or SSv4 kit?
In this case with multiple cells in a well, we recommend the SSv4 kit. The SSsc kit is ideal for single cells, i.e., a single cell per well.
Is there a protocol available for SSv4 on the mosquito HV? If so, how do I obtain this protocol?
Protocols for cDNA synthesis, library preparation, and bead clean-ups are available. Please get in touch with SPT Labtech for further assistance.
Has SSsc been used for single-cell bacterial RNA-seq?
We have not validated with bacterial cells. There was an error in the webinar; the customer has tested with Toxoplasma parasites.
Is there evidence the lysis buffer in the kit can lyse Gram-positive bacteria?
No, the provided lysis buffer has only been validated on mammalian cells. Generally, Gram-positive bacteria will require more stringent lysis conditions than those used for mammalian cells.
Which instrument was used for the SSsc miniaturization as shown in the webinar?
All liquid transfer steps were performed on mosquito HV genomics.
Have you done any validation to show if there is any cross-contamination that might occur when dispensing index primers to individual wells for library generation in a 384-well plate using the mosquito HV?
Although we have not tested index primers specifically, the positive displacement mosquito tips are designed with the elimination of cross-contamination as a central feature. We often see labs across disciplines running their mosquitos without the head cover.
To increase confidence, the tip guard on the bottom of the head cover acts as a barrier to further ensure there is no cross-contamation. Finally, tip change operations can be performed over a blank or waste plate position on the deck, eliminating any chance of cross-contamination in source or reaction wells.
What is the advantage of the mosquito system compared to the 10x system?
Plate-based, single-cell RNA-seq is a complementary approach to droplet methods, like the 10x Chromium system. It allows analysis of the full transcriptome (in contrast to 3′ counting) and detection of rare variants, isoforms, and splice variants. It is a common approach to first screen a complex population using the droplet method, followed by deep transcriptomic analysis in plates. Furthermore, it's also important to note that the mosquito system aids in increasing the throughput of plate-seq approaches, while maintaining sensitivity.
How does the SSsc kit compare to seqWell's new product, which claims improvements over SMART-Seq?
The SSsc kit maintains the highest sensitivity on the market. In a direct comparison of seqWell to Takara Bio’s SSsc kit using the challenging sample type of single PBMCs, SSsc consistently outperforms seqWell's scRNA-seq kit. You can find these data on seqWell’s website.
High-quality, full-length RNA-seq libraries from single cells (e.g., PBMCs) and nuclei.
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