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 support@takara.co.kr 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; (Pushing the limits of sensitivity for single-cell applications) 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-contamination. 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.

This kit uses oligo dT priming, so acceptable samples are Intact whole cell (1-1,000 intact cells), purified intact total (10 pg-10 ng) RNA with RIN>8. Using more than 1,000 cells for direct cDNA synthesis with SMARTer Ultra low kits is not recommended.

Even when the integrity is good, RNA can suffer from a number of impurities, including organic solvents and salts left over from extraction (e.g., phenol, chloroform), nucleases as well as other proteases that can make it through the extraction process.

Fortunately, rRNA and genomic DNA will not affect the faithful representation of in vivo gene expression levels when using the SSv4 kit.

The SSv4 improves on our previous SMARTer Ultra low chemistry and outperforms both previously published protocols (including the SMART-Seq2 method) and existing kits. The SSv4 chemistry builds on our experience from three previous generations of SMARTer Ultra low kits, and the work done by Rickard Sandberg's group at Ludwig Cancer Research on the SMART-Seq2 method.

This kit delivers the highest number of genes identified, maintains sequencing platform compatibility, and provides improved data for GC-rich transcripts from 10 pg-10 ng of total RNA(<1,000 intact cells). The SSv4 kit does this by incorporating the novel application of LNA technology used by the Ludwig team as well as innovations developed by Takara Bio.

All reagents included in the kit are to be stored at -20°C.

Yes

NucleoSpin RNA XS kit (Cat. # 740902.10) for up to 1 x 105 cultured cells. Use of carrier RNA is NOT recommended since it will interfere with oligo(dT) primed cDNA synthesis. If your RNA sample is dilute or was pre-purified using organic compounds, you may concentrate and clean up the RNA without the addition of a carrier using the NucleoSpin RNA Clean-up XS kit (Cat. # 740903.10). Traces of organic compounds (e.g., TRIzol, ethanol) in the RNA prep may interfere with reverse transcription.

It is important to collect cells using media and buffers that do not suppress cDNA synthesis. PBS buffer has been tested and is compatible with all SMARTer Ultra low kits at all inputs (1-9 μl).

PBS buffer (for 1 L; sterilize using 0.2-micron filter):
0.2 g	KCL
0.24 g	KH2PO4 (anhydrous)
8.0 g	NaCl
1.44 g	Na2HPO4 (anhydrous)
	Add H2O up to 1 L

The following media have not been tested in-house; however, they have been externally validated for use with low-input volumes (1 μl).

• SuperBlock (Pierce, Cat. # 37515)
• 1 ml of DMEM/F-12, GlutaMAX (Thermo Fisher Scientific, Cat. # 10565) + 3.6 μl of 25% BSA (Thermo Fisher Scientific, Cat. #A10008-01)

For the SSv4 kit, lysis is conducted at room temperature, while for the previous generations it is done on ice. Lyse the collected cell(s) with Reaction Buffer (Dilution Buffer + RNase Inhibitor) and incubate at room temperature for 5 minutes.

Since the Reaction Buffer contains RNase Inhibitor, we strongly recommend preparing it immediately before use. If it is not feasible to prepare the Reaction Buffer immediately before use, you may keep it on ice and add RNase Inhibitor immediately before use.

Note: Dilution Buffer contains a detergent; therefore, mix it carefully to avoid bubbles.

For cell lysis using the SSv4 kit, we recommend the addition of 1 μl of 10X Reaction Buffer followed by a 5-minute incubation at room temperature.

If you cannot immediately proceed with cDNA synthesis, you may freeze cells on dry ice and store at -80°C.

Gently centrifuge cells, remove the collection medium and freeze the cell pellets. Collected cells may also be frozen in media compatible with the SMARTer Ultra low protocol (see "What media have been tested for compatibility with direct cDNA synthesis from intact mammalian cells?").

Thaw cells immediately prior to cDNA synthesis and add Reaction Buffer containing RNase Inhibitor.

Allow cell lysis to proceed for 5 minutes at room temperature if using the SSv4.

If necessary, cells may be collected directly in Reaction Buffer containing RNase Inhibitor, followed immediately by cDNA synthesis or freezing.

Note: If cells are collected and frozen in Reaction Buffer, add fresh RNase Inhibitor after thawing cells and prior to cDNA synthesis.

In general, depending on the RNA source, integrity, input amount, and the final volume of the library, the expected yield of ds cDNA generated using SMARTer Ultra low kits is 2-17 ng. This is achieved using the optimized number of PCR cycles and ensuring cDNA amplification is in the exponential phase (i.e., avoiding overcycling). To ensure true representation of the original mRNA pool, it is critical to avoid overamplification of cDNA.

For Illumina sequencing platforms:
The SMART-Seq Library Prep Kit available in the SSv4 PLUS kit (Cat. # R400752, R400753). This kit is compatible with 1-10 ng of input cDNA generated from the SSv4 kit.
Covaris shearing followed by library construction with the ThruPLEX DNA-Seq Kit (Cat. # R400674-R400677). This kit is compatible with 50 pg-50 ng of fragmented, double-stranded DNA (<1,000 bp), allows multiplexing, and has been validated for downstream Illumina sequencing platforms.
The Nextera® XT DNA Sample Preparation Kit (Illumina, Cat. # FC-131-1024). We have found that 100-150 pg input cDNA from the SMARTer Ultra low kits gives optimal results with this sample preparation kit.

For the Ion Torrent sequencing platforms;
we recommend using the Ion Xpress Plus Fragment Library Preparation Kit (Thermo Fisher Scientific, Cat. # 4471269) and an Ion Xpress Barcode Adapter kit (Life Technologies, several Cat. #s.). This method is compatible with 1-10 ng of cDNA digested with AfaI (to remove SMART adapters) and enzymatically fragmented using reagents from the Ion Xpress Plus Fragment Library Preparation Kit.

cDNA generated with a SMARTer Ultra low kit that is sheared using Covaris technology and prepared with the Low Input Library Prep Kit typically has a size distribution of 150-600 bp with a peak at approximately 250-300 bp.

Follow the recommendations from Illumina for library pooling.

In our hands, using 100-150 pg of input cDNA with the Nextera XT DNA Sample Preparation Kit generates DNA fragments with an optimal average size for Illumina cluster generation and sequencing. Using more than 150 pg of ds cDNA is not recommended since it generates significantly larger DNA fragments, which are suboptimal for Illumina cluster generation and sequencing.

For a greater cDNA input range, we recommend using the SSv4 PLUS kit (Cat. # R400752, R400753). It allows for inputs between 1-10 ng.

No. Use 100-150 pg of ds cDNA generated with the SMARTer Ultra low kit in the input volume recommended in the Nextera XT DNA Sample Preparation Guide. Follow the rest of the protocol as written.

The Nextera kits from Illumina produce libraries with a size range of 300-1,000 bp. Please refer to the Nextera DNA Sample Preparation Guide or Nextera XT DNA Sample Preparation Guide for more specific details.

By using qPCR primers that anneal to the sequencing adaptors, you can quantify just the fraction of the library capable of cluster generation. qPCR is also extremely sensitive, consuming only a small amount of your sample and making it ideal for accurate quantification of very dilute libraries.

SMARTer kits are based on complex technology and require precise adherence to the experimental procedure. Each step of the protocol, including equipment, has been carefully optimized.

Nuclease-free thin-wall PCR tubes (0.2 ml; USA Scientific Cat. # 1402-4700) have the lowest affinity for RNA, DNA, and SPRI beads. Using strip tubes ensures better reproducibility between multiple samples and controls, and reduces the likelihood of contamination. Note: SSv4 has been validated for use with LoBind tubes (Eppendorf Cat. # 022431021).
96-well V-bottom plates (500 μl; VWR Cat. # 47743-996) recommended for some kits, enable a more efficient separation of SPRI beads from the supernatant when using large volumes of wash buffers.

[Elevated baseline in the Bioanalyzer trace]
This is commonly due to the presence of SPRI beads in the cDNA preparation. Although SPRI beads themselves do not fluoresce (nor will they bind the dye included in the Agilent High Sensitivity DNA Kit), any DNA remaining on the bead will bind dye and fluoresce.

-> To prevent bead-carryover:
- Leave the sample on the magnetic stand for an additional five minutes to attract all beads out of the solution and onto the walls of the tube.
- Remove the solution very slowly, using a long pipette tip. The smaller width of the tip allows for more distance between the beads and the tip, reducing the likelihood of disturbing the beads back into solution.

[The electropherogram exhibits a broader peak, abnormally high yield, and/or shows multiple peaks]
This usually indicates contamination. A common source of contamination is the SPRI beads, which may adsorb air pollutants (e.g., pollen).

-> To prevent contamination:
If you suspect contamination has occurred, perform a new cDNA synthesis reaction using your RNA template. Use new aliquots of SPRI beads for cDNA purification and equilibrate beads to room temperature before use.

Note: RNA from certain cell types may have high copy numbers of specific transcripts. This will result in an abnormally high peak(s) or a family of peaks on the ds cDNA electropherogram. Always perform a negative (no RNA) control to discriminate between cell-specific gene expression patterns and possible contamination.

[The electropherogram shows a broad size distribution often with multiple small peaks]
This is characteristic of a degraded RNA input sample. You may need to gather new RNA samples if you proceed with SMARTer Ultra low kits.