RNA-seq FAQs

Created specifically for single-cell and nuclei applications—and especially for cells known to have low RNA content, such as PBMCs—the SMART-Seq Single Cell Kit clearly outperforms previously published protocols (such as Smart-seq2) and existing commercial kits in terms of sensitivity and reproducibility. The improved full-length chemistry within this validated kit is associated with a higher number of useful reads and more genes detected than other popular full-length methods. The kit's plate-based workflow accepts single cells from FACS or other methods as input and uses an oligo(dT)-priming approach for first-strand cDNA synthesis.

SMART-Seq full-length chemistry has reached a new level. Read our technical note to see how you can discover more from your scRNA-seq experiments.

SMARTer Ultra low kits (including the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing) generate cDNA from 1–1,000 intact cells or 10 pg–10 ng total RNA samples. Since the SMARTer Ultra low and SMART-Seq v4 kits use oligo(dT) priming for first-strand cDNA synthesis, total RNA samples must be of high quality, with an RNA integrity number (RIN) ≥8 to ensure the availability of full-length mRNA templates required for cDNA synthesis. Ribosomal RNA (rRNA) removal or DNase treatment of RNA samples is not required for these kits. These kits selectively and efficiently amplify polyA⁺ RNA regardless of the presence of rRNA or genomic DNA.

There are several Takara Bio kits for single-cell RNA-seq that have superior performance, including the SMARTer Ultra low RNA kit for the Fluidigm C1 System for higher-throughput experiments.

The SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing improves on our previous SMARTer Ultra low kits and outperforms both previously published protocols (including the SMART-Seq2 method) and existing kits. The SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing 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 1–1,000 intact cells (or 10 pg–10 ng of total RNA). The SMART-Seq v4 kit does this by incorporating the novel application of LNA technology used by the Ludwig team as well as innovations developed by Takara Bio.

The table below indicates some of the main features of the different SMARTer Ultra low Kits for mRNA-seq.

There are slight differences in both the protocols and the composition of template-switching oligos between different SMARTer Ultra low kits. For this reason, we recommend completing your entire experiment using the same generation of SMARTer Ultra low kit.

The SMARTer Stranded RNA-Seq Kit (Cat. # 634836) is extremely sensitive and can be used with 100 pg–100 ng of full-length or degraded RNA inputs. The cDNA generated from this kit maintains strand information with >99% accuracy. This kit utilizes random priming for first-strand cDNA synthesis; therefore, total RNA samples must be rRNA-depleted or poly(A)-enriched prior to use with this kit. Random-primed cDNA synthesis is well-suited for nonpolyadenylated RNA, including noncoding RNA, bacterial RNA, and degraded RNA from FFPE and LCM samples. Illumina adapters are integrated into cDNA library preparation, reducing the workflow time to under 4 hours.

The SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian is designed for use with high-input samples (100 ng–1 µg) of mammalian total RNA of either high or low quality. Components for rRNA depletion are included alongside the core SMART technology. As with the SMARTer Stranded RNA-Seq Kit, cDNA generated from this kit maintains strand information with >99% accuracy, and Illumina adapters are incorporated during library preparation.

For 200 pg–10 ng of degraded or nonpolyadenylated RNA samples we recommend the SMARTer Universal Low Input RNA Kit for Sequencing (Cat. # 634938). This kit is compatible with low-quality total RNA (RIN 2–3) such as that obtained from LCM or FFPE samples. As with the SMARTer stranded kit, the SMARTer Universal Low Input RNA Kit for Sequencing is a random-primed kit; therefore, total RNA samples must be depleted of rRNA. The cDNA generated with this kit is compatible with either Illumina or Ion Torrent sequencing platforms.

Yes, the SMARTer smRNA-Seq Kit for Illumina was specifically designed for analysis of small non-coding RNAs ranging in size from 15–150 nt, including miRNAs, siRNAs, piRNAs, and snoRNAs.

Currently, the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing, the SMARTer Ultra Low Input RNA Kit for Sequencing - v3, and the SMARTer Universal Low Input RNA Kit for Sequencing are compatible with downstream Ion Torrent library preparation and sequencing.

SMARTer Ultra low kits utilize an oligo(dT) primer for first-strand cDNA synthesis and require 10 pg–10 ng input RNA with a RIN ≥8 to ensure selective and efficient full-length cDNA synthesis from mRNAs.

Note: DNase treatment or removal of rRNA from a total RNA prep is not required for SMARTer Ultra low kits.

Example Bioanalyzer electropherograms of RNA samples with different RINs, from highest integrity (RIN 10) to lowest integrity (RIN 2). Source: Agilent Technologies application note: RNA Integrity Number (RIN)-Standardization of RNA Quality Control.

To determine the RIN and RNA quantity we suggest using the Agilent RNA 6000 Pico Kit (Agilent, Cat. # 5067-1513). If total RNA is not limiting, you may use the Agilent RNA 6000 Nano Kit (Agilent, Cat. # 5067-1511).

• In our hands, the Agilent RNA 6000 Pico Kit was more accurate for assessing RNA quantity at lower concentrations compared to other assays tested.

Choose the most suitable RNA purification kit for your starting material (e.g., plant, tissue, mammalian cells). The RNA purification kit should be compatible with downstream cDNA synthesis and sequencing, the use of a carrier is not recommended.

Note: Some plant species have high levels of polysaccharides, which may be retained in the final RNA prep. Excess polysaccharides may block the primer from binding to the RNA template, interfering with reverse transcription.

For RNA isolation from up to 1 x 10⁵ cultured cells you may consider the NucleoSpin RNA XS kit (Cat. # 740902.10). NucleoSpin RNA XS kit specifications are provided in Table I of the NucleoSpin RNA XS Total RNA Isolation User Manual.

• The use of a poly(A) carrier during RNA purification is not recommended since it may interfere with downstream 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) as described in the NucleoSpin RNA Clean-up XS User Manual.

• Traces of organic compounds (e.g., TRIzol, ethanol) in the RNA prep may interfere with reverse transcription.

The SMARTer Universal Low Input RNA Kit for Sequencing (Cat. # 634938) has been validated for use with 200 pg–10 ng of sheared or degraded (RIN 2–3), rRNA-depleted input RNA. The optimal input RNA size distribution for this kit should peak at approximately 200 nt.

Example electropherogram of the optimal RNA size distribution for the SMARTer Universal Low Input RNA Kit for Sequencing. Human Brain Total RNA (HBR) was chemically sheared, spiked with ERCC control RNA (4 µl of a 1:1,100 dilution per 100 ng), and rRNA-depleted using a modified Ribo-Zero protocol for low-input samples. One microliter was analyzed using an Agilent 2100 Bioanalyzer (RNA 6000 Pico chip).

There are many RNA extraction and purification methods compatible with the SMARTer Universal Low Input RNA Kit for Sequencing (Cat. # 634938). When choosing a purification method, ensure that it is appropriate for the particular sample type and the amount you are working with.

Input RNA should be free of genomic or carrier DNA, and free of contaminants that would interfere with oligo-RNA template annealing or would inhibit the reverse transcriptase reaction.

Since the SMARTer Universal Low Input Kit for Sequencing (Cat. # 634938) utilizes random priming for first-strand cDNA synthesis, if rRNA is not depleted, up to 90% of sequencing reads are expected to map to rRNA.

For 10–100 ng samples of mammalian total RNA, we recommend using the RiboGone - Mammalian kit (Cat. # 634846) for rRNA depletion.

For cDNA synthesis using the SMARTer Universal Low Input RNA Kit for Sequencing (Cat. # 634938), we recommend shearing at the same time as priming the RNA. To do this, add 4 µl of 5X First-Strand Buffer to your RNA and the 3' SMART N6 CDS Primer II A (not to the Master Mix), then incubate at 94°C. For RNA with a RIN >7, incubate for 5 min. For RNA with RIN 4–7, incubate for 4 min. For RNA with a RIN of 3, incubate for 3 min. For RNA with a RIN <3, follow the protocol as directed in the SMARTer Universal Low Input RNA Kit for Sequencing User Manual.

For cDNA synthesis from intact cells, we recommend the  SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing, as this kit has the highest sensitivity in identifying the maximum number of transcripts from ultra-low inputs of RNA.

SMARTer Ultra low kits can accommodate inputs of 1–1,000 intact mammalian cells for direct cDNA synthesis.

Note: Direct cDNA synthesis from plant or insect cells has not been tested in-house with SMARTer Ultra low kits.

Electropherogram of cDNA generated from whole cells with the SMARTer Ultra Low Input RNA Kit for Sequencing - v3.

Using more than 1,000 cells for direct cDNA synthesis with SMARTer Ultra low kits is not recommended.

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):

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

Note: For the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing and the SMARTer Ultra Low Input RNA Kit for Sequencing - v3, we have only tested PBS, other media were not tested with these kits.

Refer to the user manual for the specific kit that you are using, as the lysis reaction conditions may be different for different kits. For the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing or the SMARTer Ultra Low Input RNA Kit for Sequencing - v3, 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 or on ice 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 SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing or the SMARTer Ultra Low Input RNA Kit for Sequencing - v3, we recommend the addition of 1 µl of 10X Reaction Buffer followed by a 5-minute incubation at room temperature.

For the legacy kits, the amount of Reaction Buffer will vary depending on the input volume of your RNA or cell sample. Maintaining the same volume of Reaction Buffer for all cell samples is not necessary. Do not add more than 5 µl of Reaction Buffer to your sample. If necessary, add nuclease-free water to a final volume of 10 µl. Allow lysis to proceed for 5 minutes at room temperature or on ice (4°C).

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 SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing or the SMARTer Ultra Low Input RNA Kit for Sequencing - v3. If using a legacy kit, lysis can be performed on ice (4°C) or at room temperature.

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.

ZapR is a proprietary technology that, in conjunction with R-Probes, selectively removes mammalian rRNA (18S and 28S) and human mitochondrial rRNA (m12S and m16S) sequences from the double-stranded cDNA generated by the SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian. For more details, please see the schematic in the technical note for this product, which describes the workflow of SMARTer cDNA synthesis, including ZapR-mediated removal of rRNA sequences.

R-Probes are proprietary reagents that, in conjunction with ZapR technology, facilitate the removal of mammalian rRNA (18S and 28S) and human mitochondrial rRNA (m12S and m16S) sequences from the double-stranded cDNA generated by the SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian. For more details on how R-Probes fit into the kit's workflow, see the schematic in the technical note for this product.

Please see the size distribution graph of the sequenced cDNA fragments (reflecting the sizes of captured RNAs) generated from either sheared full-length RNA or degraded RNA from FFPE tissue (RIN 2.5) in the technical note for this product.

RNA fragments as small as 100 nt are represented in the final cDNA library created with the SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian. Please see the size distribution graph of the sequenced cDNA fragments (reflecting the sizes of captured RNAs) generated from either sheared full-length RNA or degraded RNA from FFPE tissue (RIN 2.5) in the technical note for this product.

Yes, we used the SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian to generate cDNA libraries using RNA extracted from FFPE samples; please view our webinar for more information.

The cumulative size of the adapters in the SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian is 139 bp.

The final libraries may retain between 10% and 35% of rRNA sequences, depending on the RNA source. Please see the bar charts in the technical note for this product.

We have not been able to detect any off-target effects; our data show an excellent correlation between treated (with R-Probes) and untreated (without R-Probes) libraries. Please see the scatter plots in the technical note for the product.

Currently, we do not offer ZapR-mediated removal of rRNA sequences as a separate product.

No, this technology (ZapR-mediated removal of rRNA sequences) has been designed to work exclusively as an integral part of the SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian workflow.

No, R-Probes are mammalian-specific and have been validated for use with human, mouse, and rat RNA samples. R-Probes hybridizing to mitochondrial rRNA sequences (m12S and m16S) are derived from the human mitochondrial genome and are human-specific.

The duplicate rate varies depending on the RNA source, input amount, and sequencing depth, as shown in the sequencing metrics table in the technical note for this product.

In general, RNA complexity is reduced as the input amount is lowered, therefore the duplicate rate is higher.

The number of PCR cycles recommended for library amplification has been optimized depending on the initial RNA input to ensure cDNA amplification within the linear PCR amplification range. We found that the number of PCR cycles within the recommended input range does not affect the duplicate rate; rather, the duplicate rate is influenced by the input RNA amount. In general, RNA complexity is reduced as the input amount is lowered, raising the duplicate rate.

If using more than 14 PCR cycles for the amplification of an RNA-seq library with the SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian, there is a risk of overamplification of adapters. For this reason, a second purification is necessary following first-strand cDNA synthesis. This additional cleanup will remove excess oligos prior to library amplification at higher cycle numbers. If the excess oligos are not removed, they will be amplified and then sequenced with your RNA-seq library.

The SMART reaction is inherently stranded and does not require additional cDNA preparation steps to generate stranded RNA-seq data. Unlike other methods, SMART technology does not depend on AT-rich sequences for dUTP incorporation and subsequent second-strand cDNA degradation. This allows the SMARTer Stranded RNA-Seq Kit and SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian to provide strand information even for highly GC-rich genes that may lack sufficient thymidine nucleotides for dUTP incorporation.

All SMARTer stranded kits include Illumina adapters and indexes as part of the PCR primers used to amplify the cDNA.

• SMARTer Stranded RNA-Seq Kits (Cat. # 634836–634861) contain a universal forward primer (with a sequence identical to the Illumina TruSeq® Universal Adapter) and 12 reverse PCR primers for generating up to 12 uniquely indexed libraries. The indexes contained in the 12 reverse primers correspond to those in the Illumina TruSeq DNA LT Sample Prep Kit (adapters AD001–AD012).
• The PCR primers included in the SMARTer Stranded RNA-Seq Kit HT (Cat. # 634862) contain indexes identical to those found in the Illumina TruSeq DNA HT Sample Prep Kit. The 8 forward primers contain indexes identical to D501–D508, and the 12 reverse primers contain indexes identical to D701–D712.

The SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian (Cat. # 634873–634878) includes different primer sets depending on the reaction size. The indexes included are identical to those in the Illumina TruSeq DNA HT Sample Prep Kit.

• The 12- and 24-reaction kits (Cat. # 634873, 634874) include one forward primer (with an index identical to D502) and 12 reverse primers (with indexes identical to D701–D712)
• The 48-reaction kit (Cat. # 634875) includes 4 forward primers (with indexes identical to D501–D504) and 12 reverse primers (with indexes identical to D701–D712)
• The 96–480 reaction kits (Cat. # 634876–634878) include 8 forward primers (with indexes identical to D501–D508) and 12 reverse primers (with indexes identical to D701–D712)

The nucleotide sequences for the different indexes can be found in the corresponding user manual.

SMARTer stranded cDNA libraries include Illumina adapters; not all indexes can be pooled in order to maintain enough nucleotide diversity for sequencing. Follow Illumina recommendations (e.g., in the "TruSeq DNA Sample Preparation Guide") for pooling libraries.

We recommend analyzing double-stranded (ds) cDNA generated with SMARTer kits using an Agilent 2100 Bioanalyzer and the High Sensitivity DNA Chip (Agilent, Cat. # 5067-4626) before sequencing.

Prior to cDNA library analysis, ensure that the electropherogram of the High Sensitivity DNA Ladder is properly displayed: showing a flat baseline, well-resolved ladder peaks, and properly identified Lower and Upper Markers. If the High Sensitivity DNA Ladder electropherogram shows an unexpected pattern, consult the Agilent 2100 Bioanalyzer Expert Maintenance and Troubleshooting Guide.

Electropherogram of the Agilent High Sensitivity DNA Ladder showing the flat baseline, well-resolved ladder peaks, and the properly identified Lower (43.00) and Upper (113.00) Markers.

SMARTer Ultra low kits: Successful cDNA synthesis and amplification should produce a cDNA library spanning 400–9,000 bp. The main peak should occur at approximately 2,000 bp.

SMARTer stranded kits: Successful cDNA synthesis and amplification with the SMARTer Stranded RNA-Seq Kit should yield a distinct Bioanalyzer electropherogram peak spanning 150–1,000 bp, centered on approximately 300 bp. When using the SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian, successful synthesis should yield a distinct peak spanning 200–1,000 bp, centered on approximately 300 bp.

The SMARTer Universal Low Input RNA Kit for Sequencing: Successful cDNA synthesis and amplification should yield a distinct Bioanalyzer electropherogram peak spanning 100–1,000 bp, centered on approximately 200 bp.

Note: The cDNA library should be representative of the full-length mRNA distribution which may differ between different tissues or cell types.

SMARTer Ultra low kits

The cDNA yield in newer generations of SMARTer Ultra low kits is higher compared to that of the legacy kits. Please refer to the user manual of your specific kit for more details. 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. See "Cycling Guidelines Based on Amount of Starting Material" in the user manual for your particular SMARTer Ultra low kit.

Electropherogram of cDNA libraries generated with different amounts of Mouse Brain Total RNA (including a no RNA template control; NTC). Depending on the initial RNA input, optimization of PCR cycle number may be required to ensure a yield of 2–17 ng of cDNA.

SMARTer stranded kits

The SMARTer Stranded RNA-Seq Kit generates RNA-seq libraries for Illumina sequencing at a final concentration >7.5 nM. The SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian generates RNA-seq libraries for Illumina sequencing at a final concentration of 2–10 ng/µl.

SMARTer Universal Low Input RNA Kit for Sequencing

The expected yield of ds cDNA generated using the SMARTer Universal Low Input RNA Kit for Sequencing is 2–10 ng.

Estimate the yield of ds cDNA using an Agilent 2100 Bioanalyzer:

1. Open the ds cDNA electropherogram using Agilent 2100 Expert Software.
2. Choose the "Region Table" tab.
3. Select the expected size range of ds cDNA appropriate for your SMARTer kit. The ds cDNA concentration will be displayed below the graph.
   1. For SMARTer Ultra low kits: select the region encompassing 400–9,000 bp.
   2. For the SMARTer stranded kit: select the region encompassing 150–1,000 bp.
   3. For the SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian: select the region encompassing 200–1,000 bp.
   4. For the SMARTer universal kit: select the region encompassing 100–1,000 bp.
4. To estimate the total amount of ds cDNA, multiply the ds cDNA concentration (pg/µl or pmol/l) by the volume (µl) of the ds cDNA sample (taking any dilution factor into account).

Evaluating ds cDNA concentration using Agilent 2100 Expert Software. The "Region Table" tab is indicated by the green arrow, the selected region is indicated by the blue vertical bars. The cDNA concentration is indicated by the red arrow. cDNA was generated using either 100 pg of Mouse Brain Total RNA spiked with ERCC as input for the SMARTer Ultra Low Input RNA Kit for Sequencing - v3 (Panel A), the SMARTer Stranded RNA-Seq Kit with 1 ng of poly(A)-enriched Human Brain Total RNA (Panel B), the SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian with 1 µg of Control Mouse Liver Total RNA (Panel C), or the SMARTer Universal Low Input RNA Kit for Sequencing with 2 ng of chemically fragmented Human Brain Total RNA (Panel D).

If the ds cDNA yield is less than 2 ng, you may further amplify the ds cDNA using several additional PCR cycles, based on the ds cDNA concentration (determined by Agilent 2100 Expert Software). Continue to avoid overcycling. It is preferable to use too few cycles rather than too many.



An electropherogram trace of low concentration ds cDNA generated with a SMARTer Ultra low kit. The red arrow indicates the concentration as determined by Agilent 2100 Expert Software. This cDNA can be further amplified using 1–2 additional PCR cycles.

PCR-amplified ds cDNA can be analyzed directly from the PCR reaction mix, prior to SPRI bead purification, using an Agilent 2100 Bioanalyzer. The ds cDNA profile will contain a large peak immediately following the Lower Marker; this represents the primer or primer dimers. The Bioanalyzer software may assign the primer/primer-dimer peak as the Lower Marker. If this occurs, manually reassign the Lower Marker.

If the ds cDNA yield is low, you may further amplify the cDNA, using several additional PCR cycles, before continuing with purification with SPRI beads as described in the protocol.

Note: If you are using a kit that includes a SPRI bead purification step prior to PCR amplification in the protocol, pipette the cDNA sample carefully to ensure that SPRI beads are not introduced into the Agilent 2100 Bioanalyzer.

Electropherograms of unpurified, PCR-amplified DNA. Panel A shows a negative control, and Panel B shows a positive control generated with 15 cycles of PCR. The green arrow indicates the primer/primer-dimer peak.

We recommend two preparation methods for Illumina sequencing platforms:

• 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 platform, 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.

Note: Ion Torrent library preparation is only compatible with cDNA generated using the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing or the SMARTer Ultra Low Input RNA Kit for Sequencing - v3.

Covaris-sheared ds cDNA should span 100–500 bp with a peak of approximately 200 bp. To ensure optimal Covaris ds cDNA shearing:

• Do not load more than 75 µl of ds cDNA per 100 µl Covaris tube.
• Avoid introducing air bubbles when loading the ds cDNA in the Covaris tube.

Example Bioanalyzer electropherogram of Covaris-sheared ds cDNA. Recommendations for Covaris DNA shearing conditions can be found in SMARTer Ultra low kit user manuals.

Covaris shearing parameters, provided in the user manuals of the SMARTer Ultra low kits, were optimized using a Covaris S220 Focused-ultrasonicator.

• If you are using another type of Covaris apparatus, please consult the manufacturers for the recommended parameters to ensure DNA is in the size range of 100–500 bp with a peak at approximately 200 bp.

The intensity for the S220 Covaris protocol is an equivalent to Peak Incident Power (W) set at 175.

The ThruPLEX DNA-Seq Kits (Cat. # R400674–R400677) have the following advanced features:

• High performance from low inputs—generate libraries from 50 pg to 50 ng of input material 
• Fast and simple workflow—three-step, single-tube or single-well protocol takes only about two hours, no transfers necessary 
• Compatible with major target enrichment platforms—suitable for use with Agilent SureSelect, Roche Nimblegen SeqCap EZ, and IDT xGen Lockdown probes

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.


Example Bioanalyzer electropherogram of a cDNA library prepared for Illumina sequencing. The cDNA library was prepared from 10 pg of Mouse Brain Total RNA using a SMARTer Ultra low kit and the Low Input Library Prep Kit.

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.



Example Bioanalyzer electropherograms of RNA-seq libraries generated from either 130 pg (Panel A) or 1 ng (Panel B) of cDNA generated using a SMARTer Ultra low kit. 130 pg of input cDNA generates libraries of optimal size for Illumina cluster generation and sequencing.

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.



Example Bioanalyzer electropherograms of cDNA libraries prepared for Illumina sequencing from 1 ng of Mouse Brain Total RNA (Panel A) or 1 ng of Human Universal Total RNA (Panel B) using a SMARTer Ultra low kit with the Nextera DNA Sample Preparation Kit and the modified Nextera protocol provided by Takara Bio. 5 ng of ds cDNA and 1 µl of Tagment DNA Enzyme (TDE1) were used for both samples. The difference in ds cDNA yield is related to the yield and size distribution occurring during initial cDNA synthesis, which may vary for different RNA sources. This will, in turn, result in different ds cDNA fragmentation patterns and yields.

To obtain the highest quality NGS data, loading the flow cell with an appropriate amount of library DNA is essential. An insufficient amount of library DNA will generate low-density clusters and reduced sequencing yield. Excessive amounts of library DNA may increase cluster density, resulting in poor data quality. In addition, for multiplexed sequencing, there must be an equal amount of each library in a pool to obtain a uniform number of reads across libraries. For libraries prepared for Illumina sequencing, we recommend the Library Quantification Kit.

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.

qPCR only measures the library molecules that can be used for cluster generation. Other methods cannot differentiate between DNA molecules with or without adaptors, resulting in inaccurate quantification of the functional fraction of the library.

A positive-control cDNA synthesis reaction, using control RNA included in each SMARTer kit, enables verification of kit performance and components and helps in evaluation of your sample cDNA library.

Tips for preparing the control reactions:

• Prepare fresh dilutions of the Control RNA. Do not use previously diluted low-concentration RNA samples, since RNA is less stable at low concentrations.
• If attempting to use previously diluted Control RNA, analyze its integrity using an Agilent Bioanalyzer 2100.
• Prepare Control RNA dilutions in nuclease-free water or Reaction Buffer containing fresh RNase Inhibitor.
• Use nuclease-free, nonsticky 1.5-ml tubes.
• Avoid pipetting small volumes (1 µl or less). Dilutions of the Control RNA will be more accurate if, after the first 1-µl dilution, subsequent dilutions are performed using larger volumes (4–5 µl) of RNA.

A negative control (performing the entire cDNA synthesis and purification procedure in the absence of any RNA input, but maintaining the same reaction volume) is essential for the evaluation of cDNA synthesis as well as for identifying potential problems, including contamination. We recommend performing a negative control reaction each time the protocol is performed, especially when using the lowest input RNA concentrations.

The following magnetic devices, recommended in the user manuals for some SMARTer cDNA synthesis kits, have been validated for SPRI bead-based purification of cDNA in different types of tubes. You may also make your own magnetic separator using rare earth magnets.

For nuclease-free thin-wall PCR tubes (0.2 ml; USA Scientific, Cat. # 1402-4700):

• SMARTer-Seq Magnetic Separator - PCR Strip, Cat. #635011

For 96-well V-bottom plates (500 µl; VWR, Cat. # 47743-996):

• Magnetic Stand-96 (Thermo Fisher Scientific, Part # AM10027)

Using two devices ensures optimal separation of SPRI beads from the supernatant and avoids magnetic bead contamination of the cDNA prep, which may result in a distorted Bioanalyzer electropherogram. A very important reason to use two separate magnetic stands is to avoid cross-contamination in kits that require two bead-purification steps. One separation device should be located in the PCR Clean Work Station, while a second magnetic separation device should be located in the General Lab. 

To ensure that purification of cDNA using SPRI beads occurs efficiently throughout the protocol, use the magnetic device specifically recommended for each type of tube. If the protocol requires multiple purifications, do not use the same magnetic device for all steps.

• Aliquot SPRI beads prior to use to avoid cross-contamination.
• Bring SPRI-bead aliquots to room temperature prior to purification to facilitate binding of cDNA, and to decrease the possibility of contamination with air pollutants. Cold SPRI beads have a higher adsorption capacity for air contaminants such as pollen.
• Mix SPRI beads with the sample by thorough pipetting. Do not vortex the beads once they are added to the samples. Vortexing can shear the DNA or break it away from the beads.
• For kits requiring purification prior to PCR amplification, ensure complete removal of the reverse transcription reaction mixture from the bead-bound first-strand cDNA. Residual reverse transcription reaction mixture may interfere with downstream PCR amplification.
• Ensure that SPRI beads are completely removed from the PCR-amplified double-stranded cDNA.
• Properly dry the SPRI bead pellet after washing; overly dry pellets may affect the DNA elution efficiency. Click here to see how the ideal bead pellet looks.

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: The SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing and the SMARTer Ultra Low Input RNA Kit for Sequencing - v3 have also 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.

1. 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.

Electropherograms of magnetic bead-contaminated cDNA sample (Panel A) and the same sample with properly removed magnetic beads (Panel B).

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.

2. 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).

Electropherogram of cDNA contaminated with pollen from the SPRI beads.

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.



An electropherogram of cDNA generated from degraded RNA template. The broad size distribution and multiple small peaks indicate poor-quality cDNA generated using the SMARTer Ultra low kits with degraded input RNA.

All stranded kits:

Few reads from the sequencing run, or few clusters passing filter. SMARTer stranded libraries can have a lower than average pass-filter rate due to low complexity for the first three cycles. Illumina software has problems interpreting low complexity libraries. Decreasing the cDNA library loading concentration and/or spiking in 5–10% PhiX control DNA (Illumina) may correct this issue.

SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian:

Background in the no-RNA control. Background arises due to amplification of environmental contaminants in the reagents and is enhanced by PCR protocols that involve higher amplification cycle numbers. RNA control background will not be visible if performing up to 13–14 cycles in PCR2 of the workflow (typically, no background is detected for up to 18 total PCR cycles). If performing 16 cycles in PCR2, the total number of PCR cycles will be 21.

Few reads from the sequencing run, or few clusters passing filter. SMARTer universal libraries can have a lower than average pass-filter rate (%PF) due to very low library complexity for the first two cycles followed by low complexity for the next five cycles, before the complexity of bases becomes random. Illumina software has problems interpreting low complexity libraries. Decreasing the cDNA library loading concentration and/or spiking in 5–10% PhiX control DNA (Illumina) may correct this issue.

ZapR is a proprietary technology that, in conjunction with R-Probes, selectively removes mammalian rRNA (18S and 28S) and human mitochondrial rRNA (m12S and m16S) sequences from double-stranded cDNA generated by the following kits:

• SMARTer Stranded Total RNA-seq Kit v2 - Pico Input Mammalian
• SMART-Seq Stranded Kit
• SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian

For more information, please see the boxed section of the following figure from our technical note, Stranded libraries from picogram-input total RNA (v2). In this part of the workflow, ribosomal cDNA (originating from rRNA) is cleaved by ZapR in the presence of R-Probes. Library fragments originating from non-rRNA molecules remain untouched, and are enriched via the second round of PCR amplification. 

Schematic of technology in the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian. SMART technology is used in this ligation-free protocol to preserve strand-of-origin information. Random priming (represented as the green N6 Primer) allows the generation of cDNA from all RNA fragments in the sample, including rRNA. When the SMARTScribe Reverse Transcriptase (RT) reaches the 5' end of the RNA fragment, the enzyme's terminal transferase activity adds a few non-templated nucleotides to the 3' end of the cDNA (shown as Xs). The carefully designed Pico v2 SMART Adapter (included in the SMART TSO Mix v2) base-pairs with the non-templated nucleotide stretch, creating an extended template to enable the RT to continue replicating to the end of the oligonucleotide. The resulting cDNA contains sequences derived from the random primer and the Pico v2 SMART Adapter used in the reverse transcription reaction. In the next step, a first round of PCR amplification (PCR1) adds full-length Illumina adapters, including barcodes. The 5' PCR Primer binds to the Pico v2 SMART Adapter sequence (light purple), while the 3' PCR Primer binds to sequence associated with the random primer (green). The ribosomal cDNA (originating from rRNA) is then cleaved by ZapR v2 in the presence of the mammalian-specific R-Probes v2. This process leaves the library fragments originating from non-rRNA molecules untouched, with priming sites available on both 5' and 3' ends for further PCR amplification. These fragments are enriched via a second round of PCR amplification (PCR2) using primers universal to all libraries. The final library contains sequences allowing clustering on any Illumina flow cell.

R-probes target mammalian rRNA (18S and 28S) and human mitochondrial rRNA (m12S and m16S) sequences only. R-probes do not target 5S and 5.8S rRNAs or the 45S rRNA precursor. ZapR will only remove human mitochondrial rRNAs; mitochondrial rRNAs from non-human mammalian species will not be removed.

No, ZapR-mediated removal of rRNA sequences has been designed to work exclusively as an integral part of the workflows of the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian, SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian, and SMART-Seq Stranded kits.

R-Probes v2, ZapR v2, and ZapR Buffer (10X) are not available separately and are only included as components of the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian, SMART-Seq Stranded Kit, and SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian.

ZapR-mediated removal of rRNA sequences may vary depending on RNA source and integrity. Compromised RNA may retain a higher percent of rRNA sequences in the final cDNA library. Depending on RNA source, larger input quantities (e.g., 50 ng) may contribute to a higher percentage of rRNA sequences in the final SMARTer Stranded v2 - Pico cDNA library. Lower input quantities (e.g., 0.1 ng) result in a higher duplication rate due to low diversity of the input RNA sample.

Examples of the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian sequencing metrics are presented below:

The SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian is compatible with purified RNA obtained from FFPE, PFA-fixed, and LCM cells, with the following caveats:

• RNA from FFPE or PFA-fixed cells must be decrosslinked. Due to the modification of nucleotides by the FFPE and PFA fixation processes, only ~10% of the input RNA may be available for reverse transcription and cDNA synthesis. We therefore recommend using >5 ng for cDNA synthesis with this kit.
• Do not use any carrier or coprecipitant during RNA purification, as it may give inaccurate RNA concentration readings and interfere with cDNA synthesis.
• Do not use purification kits that omit DNAse treatment, as FFPE, PFA-fixed, and LCM cells often contain large quantities of single-stranded DNA that can act as a template for reverse transcriptase and contaminate the final cDNA library.

Therefore, if using RNA purified from FFPE, PFA-fixed, or LCM samples, we highly recommend additional DNase treatment in the RNA eluate (in addition to any on-column DNase treatment) as described in the following:

• NucleoSpin RNA XS Total RNA Isolation User Manual (Section 5.4, "rDNase digestion in the eluate")
• NucleoSpin totalRNA FFPE - FFPE XS RNA Isolation User Manual (Section 5.3, "DNA digestion in the RNA eluates")

See our technical note, Stranded libraries from FFPE inputs (v2), for more information.

The SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian is not compatible with cDNA synthesis directly from FFPE, PFA-fixed, or LCM cells.

This kit is compatible with purified RNA obtained from FFPE, PFA-fixed, and LCM cells, with the following caveats:

• RNA from FFPE and PFA-fixed cells must be decrosslinked. Due to the modification of nucleotides by the FFPE and PFA fixation processes, only ~10% of the input RNA may be available for reverse transcription and cDNA synthesis. We therefore recommend using >5 ng for cDNA synthesis with this kit.
• Do not use any carrier or coprecipitant during RNA purification, as it may give inaccurate RNA concentration readings and interfere with cDNA synthesis.
• Do not use purification kits that omit DNAse treatment, as FFPE, PFA-fixed, and LCM cells often contain large quantities of single-stranded DNA that can act as a template for reverse transcriptase and contaminate the final cDNA library.

Therefore, if using RNA purified from FFPE, PFA-fixed, or LCM samples, we highly recommend additional DNase treatment in the RNA eluate (in addition to any on-column DNase treatment) as described in the following:

• NucleoSpin RNA XS Total RNA Isolation User Manual (Section 5.4, "rDNase digestion in the eluate")
• NucleoSpin totalRNA FFPE - FFPE XS RNA Isolation User Manual (Section 5.3, "DNA digestion in the RNA eluates")

See our technical note, Stranded libraries from FFPE inputs (v2), for more information.

This kit is not compatible with cells or RNA stored in RNAlater (Thermo Fisher), as this storage solution denatures proteins. RNA from cells stored in RNAlater must be purified to be used as input for the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian cDNA synthesis.

The SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian has been validated with inputs of 50–1,000 whole, undamaged cells. Damaged cells are not suitable as a direct input due to the fact they usually contain high quantities of single-stranded DNA, which can act as a template for reverse transcription and contaminate the final cDNA library. The protocol for cell inputs is available upon request.

For cDNA synthesis directly from cells, it is essential that the cells remain undamaged during cell collection, as damaged cells may contain compromised DNA that can act as a template for reverse transcriptase and contaminate the final cDNA library.

If feasible, we recommend verification of cell integrity prior to FACS collection. Please note that the low-pressure setting should be used on the flow sorting system and that the stream carrying cells should be aimed at the center of the bottom of a prechilled collection well/tube.

We also recommend a preliminary cell sorting pilot study: sort 50, 100, and 1,000 cells using the appropriate nozzle and pressure settings to determine the transfer volume of the sheath fluid. If the actual sheath fluid transfer volume varies from the predicted transfer volume, decrease the volume of nuclease-free water used to prepare the cell collection buffer. See additional information in the "Supplementary protocol for processing intact-cell inputs with SMARTer Pico v2," available on request.

Potentially. The collected cell volume will depend on the cell diameter, so we recommend performing a preliminary cell sorting pilot study: sort 50, 100, and 1,000 cells using the appropriate nozzle and pressure settings to determine the transfer volume of the sheath fluid. If this varies from the predicted transfer volume, you may wish to adjust the volume of cell collection buffer.

We recommend the NucleoSpin RNA XS and NucleoSpin totalRNA FFPE XS kits for purification of RNA for cDNA synthesis.

Due to the high sensitivity of the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian, all remnants of DNA should be removed using DNase treatment. This is due to the fact that single-stranded DNA generated during the fragmentation step can be randomly primed and act as a template for reverse transcription, thus contaminating the final cDNA library. To ensure complete removal of DNA, we recommend the above Macherey-Nagel kits as they offer the option of an additional rDNase digestion step of the purified RNA eluate.

For more information, please review the following:

• NucleoSpin RNA XS Total RNA Isolation User Manual (Section 5.4, "rDNase digestion in the eluate")
• NucleoSpin totalRNA FFPE - FFPE XS RNA Isolation User Manual (Section 5.3, "DNA digestion in the RNA eluates")

Please note that Macherey-Nagel nucleic acid purification products are only available from Takara Bio in North America, India, and Japan.

We recommend RNA inputs >100 nt.

Additional data (presented below) were generated for the technical note, Stranded libraries from picogram-input total RNA (v1). While this data was generated using the v1 chemistry, it is also applicable to the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian.

RNA is heat-fragmented in the presence of Mg²⁺ (included in the 5X First-Strand Buffer). Fragmentation is then stopped by placing the reaction mixture on ice for 2 minutes.

The 72°C incubation step is performed in the absence of Mg²⁺. This avoids fragmentation but permits the relaxation of RNA secondary structure and random primer annealing.

Control Total RNA, included in the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian, SMART-Seq Stranded Kit, and SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian kits, is Human Brain Total RNA.

Libraries generated with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian do not require the inclusion of extra PhiX DNA beyond the typical 1% recommended by Illumina. If you are performing paired-end sequencing on a two-channel Illumina sequencing instrument, 5–10% PhiX DNA may be added.

Since the indexing primers included with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian are incorporated via PCR carrying a proprietary overlap with the first-strand cDNA template, other vendors' indexing primers are not compatible with this kit. If you need additional indexes beyond those included with the kit, we recommend our SMARTer RNA unique dual index kits.

The final cDNA library generated with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian carries Illumina TruSeq® HT (currently known as TruSeq CD) indexing primers. The nucleotide sequences of the corresponding indexes are provided in the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian User Manual under the table titled, "Indexing Primer Set HT for Illumina v2 adapter sequences." 

The complete nucleotide sequences of the corresponding Illumina TruSeq CD (HT) adapters are available from Illumina.

The final cDNA library generated with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian carries Illumina TruSeq® HT (currently known as TruSeq CD) indexing primers. The nucleotide sequences of the TruSeq HT i5/i7 indexes are available in the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian User Manual. The complete nucleotide sequences of the corresponding Illumina TruSeq HT adapters are available from Illumina.

After the demultiplexing of Illumina adapters, the only nontemplated nucleotides required to be trimmed prior to mapping are the 3 nt in Read2, as described in Appendix A of the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian User Manual. There are no additional adapter sequences in the final cDNA library. The N6 Random Primer carries an overlap with the Illumina adapter and anneals to the complementary sequence of the RNA template. The random hexamer sequence is part of the RNA template, while the overlap with the Illumina adapter will be demultiplexed as part of the Illumina indexing primer.

The SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian has a slight 5' end bias, which is common for randomly primed libraries (Figure 1). 

Figure 1. Gene body coverage of libraries generated with the SMARTer Total RNA-Seq Kit v2 - Pico Input Mammalian.

The duplication rate depends on several factors including the input cell/RNA complexity and the sequencing depth. For instance, low RNA and cell inputs have lower complexity, which results in higher duplication rates in the final cDNA libraries. Low-quality RNA inputs and high sequencing depth may also result in a higher duplication rate. Sample sequencing metrics are provided below from our webinar, "A new method for SMARTer library preparation from challenging RNA samples," and our technical note, "Stranded libraries from picogram-input total RNA (v2)."

The SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian uses a random priming strategy that generates cDNA from coding, noncoding, and alternatively spliced RNAs that may carry introns. Additionally, previously unannotated transcripts may be assigned as introns by your software's default.

Cell and RNA inputs show good correlation (all Pearson correlation coefficients >0.91) of sequencing metrics with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian. Additional details, including sequencing metric data, are provided below from our webinar, A new method for SMARTer library preparation from challenging RNA samples:

In our technical note, Stranded libraries from picogram-input total RNA (v2), we demonstrated superior sequencing performance of the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Pico v2) compared to the v1 chemistry (see below). Additionally, an independent comparison revealed that our Pico v2 kit demonstrates superior transcript detection, expression profiling efficiency, uniquely mapped raw read counts, and percentage of uniquely mapped reads versus two competitors.

The advantages of the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian are:

• No low diversity in Read1 (Figure 1) due to the altered position of Read1 primer, which does not read through the GC-rich nucleotides of the SMART adapter (depicted as a triple X in Figure 2)
• Improved efficiency of ZapR removal of rRNA sequences (compare proportion of rRNA reads with each chemistry; shaded row in Table I)
• Validated with SeqAmp CB PCR Buffer, which reduces the viscosity of the SeqAmp DNA Polymerase reaction mix and facilitates AMPure bead purification (Figure 3)
• Broad input quantity range (Figure 4): the SMARTer Stranded Total RNA-Seq Kit - Pico v2 has been validated with 0.1–50 ng of total RNA. Lower RNA input quantities may generate higher duplicate rates due to lower RNA complexity. Depending on the RNA source, RNA inputs >10 ng may contribute to a higher percentage of rRNA in the final SMARTer Stranded v2 Pico cDNA library.
• Improved performance with cell-free RNA (Figure 5)
• Compatible with cDNA synthesis directly from 50–1,000 whole, undamaged cells (Figure 6; protocol available upon request from technical support)

Figure 1. Improved pass-filter rates (%PF) with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian. Libraries generated with the Pico v1 or Pico v2 kits were pooled and run on NextSeq® 500 or MiniSeq™ instruments, as indicated. For each graph, blue boxplots indicate the distribution of cluster densities for unfiltered (i.e., raw) reads, while the green boxplots indicate the distribution of cluster densities for reads that passed filtering. Quantities of reads passing filter (in millions) and %PF values for each sequencing run are included above each graph. The expected number of reads passing filter according to Illumina specifications was 130 million reads for runs on the NextSeq and 25 million reads for runs on the MiniSeq. Proportions of reads that aligned to PhiX sequences ranged from 0.5% to 1.15% for all sequencing runs. As indicated in the graphs, libraries generated with the Pico v2 kit achieved higher %PF values for both Illumina platforms relative to libraries generated with the Pico v1 kit, and yielded quantities of reads passing filter that greatly exceeded the Illumina specifications.

Figure 2. Structural features of final libraries generated with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian. The adapters added using 5' PCR Primer HT and 3' PCR Primer HT contain sequences allowing clustering on any Illumina flow cell (P7 shown in light blue, P5 shown in red), Illumina TruSeq® HT indexes (Index 1 [i7] sequence shown in orange and Index 2 [i5] sequence shown in yellow), as well as the regions recognized by sequencing primers Read Primer 2 (Read 2, purple) and Read Primer 1 (Read 1, green). Read 1 generates sequences antisense to the original RNA, while Read 2 yields sequences sense to the original RNA (orientation of original RNA denoted by 5' and 3' in dark blue). The first three nucleotides of the second sequencing read (Read 2) are derived from the Pico v2 SMART Adapter (shown as Xs). These three nucleotides must be trimmed prior to mapping if performing paired-end sequencing.

Figure 3. Improved bead-pellet formation with new SeqAmp CB PCR Buffer. The PCR buffer included in the Pico v2 kit was re-formulated to allow for faster, tighter bead-pellet formation. Following magnetic separation for a fixed period, bead pellets formed in the new SeqAmp CB PCR Buffer included in the Pico v2 kit (right) are tighter than those formed in the original PCR buffer included in the Pico v1 kit (left). Tighter bead pellets tend to dry more evenly and are easier to resuspend than pellets that are broader and more diffuse.

Figure 4. Sequencing metrics for human versus mouse RNA samples. Depending on RNA source, larger input quantities (50 ng) may contribute to a higher percentage of rRNA sequences in the final SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian cDNA library. Lower input RNA quantities (e.g., 0.1 ng) result in a higher duplication rate due to the low diversity of the input RNA sample.

Figure 5. Improved cell-free RNA-seq libraries with the Pico v2 chemistry versus the Pico v1. In a direct comparison of the Pico v2 versus v1 chemistries, the Pico v2 demonstrated higher mapping rates and more genes detected at 150-pg inputs, while lower quantities of ribosomal and mitochondrial reads were observed regardless of input mass.

Figure 6. Comparable data between cells and purified RNA with the Pico v2 kit. Sequencing metrics taken from cells and purified RNA were highly reproducible, with a similar number of transcripts identified between 1,000 cells and RNA extracted from 1,000 cells.

Additional information on these advantages are presented below with data taken from the webinar, A new method for SMARTer library preparation from challenging samples, and the technical note, Stranded libraries from picogram-input total RNA (v2).

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).

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 platform, 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.