Pushing the limits of sensitivity for single-cell applications Q&A

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.

The SSsc library preparation kit has been miniaturized to 1/2 volume. Please contact technical_support@takarabio.com for more information.

With the SSsc PLUS kit, 96 samples can be multiplexed in a single sequencing run.

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 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.

Protocols for cDNA synthesis, library preparation, and bead clean-ups are available. Please get in touch with SPT Labtech for further assistance.

We have not validated with bacterial cells. There was an error in the webinar; the customer has tested with Toxoplasma parasites.

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.

All liquid transfer steps were performed on mosquito HV genomics.

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.

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.

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.