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RNA-seq FAQs

What kit is right for my application?

For whole cells or ultra-low-input total RNA samples: SMARTer Ultra Low kits (dT-primed)

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 kits and SMART-Seq v4 kit 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 Clontech 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.

What are the benefits of using the fourth-generation SMART-Seq v4 kit versus other ultra-low input mRNA-seq kits?

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 improved data from 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 at Clontech.

How do the various generations of ultra-low input mRNA-seq kits compare in terms of features and product components?

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

  SMARTer Ultra Low RNA Kit for Illumina Sequencing SMARTer Ultra Low Input RNA for Illumina Sequencing - HV kit SMARTer Ultra Low Input RNA Kit for Sequencing - v3 SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing
Sample Input Volume 1 µl Up to 9 µl Up to 9 µl Up to 10.5 µl
Sample Input 1–1000 intact cells
10 pg–10 ng total RNA
PCR Polymerase Advantage 2 DNA Polymerase SeqAmp DNA Polymerase
Reverse Transcriptase SMARTScribe Reverse Transcriptase
Template-switching oligo Continuously improved, proprietary SMART oligo (each generation of SMARTer Ultra Low kits includes modifications that improve the efficiency of template switching)
Components storage conditions Majority of reagents stored –20°C; template-switching oligo stored at –80°C All reagents stored at –20°C

I am currently using a legacy SMARTer Ultra Low kit, can I switch to the SMART-Seq v4 kit?

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.

For full-length or degraded total RNA with strand information maintained: SMARTer Stranded kits (random primed)

The SMARTer Stranded RNA-Seq Kit (Cat. No. 634836) is extremely sensitive and can be used with 100 pg–100 ng full-length or degraded RNA samples. 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 non-polyadenylated RNA, including non-coding RNA, bacterial RNA, and degraded RNA from FFPE and LCM samples. Illumina® adapters are integrated into cDNA library preparation, this reduces 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 to 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 integrates Illumina adapters into sequencing library preparation.

For degraded total RNA samples: SMARTer Universal Low Input RNA Kit for Sequencing (random-primed)

For 200 pg–10 ng degraded or non-polyadenylated RNA samples we recommend the SMARTer Universal Low Input RNA Kit for Sequencing (Cat. No. 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.

Can I use SMARTer kits for the analysis of mature miRNAs?

No current SMARTer kit is recommended for the analysis of mature miRNAs.

How should I prepare RNA samples for cDNA synthesis?

SMARTer Ultra Low kits:

What are the requirements for RNA quality and quantity when using SMARTer Ultra Low kits?

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 electropherograms used to train the RIN software (Agilent Technologies)

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.

What methods can I use to assess RNA quality and quantity?

To determine the RIN and RNA quantity we suggest using the Agilent RNA 6000 Pico Kit (Agilent, Cat. No. 5067-1513). If total RNA is not limiting you may use the Agilent RNA 6000 Nano Kit (Agilent, Cat. No. 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.

What RNA purification kits are recommended?

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 a high levels of polysaccharides, which may be retained in the final RNA prep. The excess polysaccharides may block the primer from binding to the RNA template, interfering with the reverse transcription.

For RNA isolation from up to 1 x 105 cultured cells you may consider the NucleoSpin RNA XS kit (Cat. No 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. No. 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.

SMARTer Universal kit:

What are the requirements for RNA quantity and quality when using the SMARTer Universal Low Input RNA Kit for Sequencing?

The SMARTer Universal Low Input RNA Kit for Sequencing (Cat. No. 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.

rRNA-depleted input for the SMARTer Universal kit

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).

What RNA purification kits are compatible with the SMARTer Universal kit?

There are many RNA extraction and purification methods compatible with the SMARTer Universal Low Input RNA Kit for Sequencing (Cat. No. 634938). When choosing a purification method, ensure that it is appropriate for the particular sample type and 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.

Why do I need to remove ribosomal RNA?

Since the SMARTer Universal Low Input Kit for Sequencing (Cat. No. 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.

What ribosomal RNA depletion methods do you recommend?

For 10–100 ng samples of mammalian total RNA, we recommend using the RiboGone - Mammalian kit (Cat. No. 634846) for rRNA depletion.

How do I shear rRNA-depleted RNA?

For cDNA synthesis using the SMARTer Universal Low Input RNA Kit for Sequencing (Cat. No. 634938), we recommend shearing at the same time as priming the RNA. To do this add 4 µl 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 a 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.

FAQs for cDNA synthesis directly from cells:

Which SMARTer Ultra Low kit is recommended for direct cDNA synthesis from whole mammalian cells?

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.

How many cells can I use for direct cDNA synthesis with SMARTer Ultra Low kits?

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.

Electropherograms of amplified SMARTer cDNA

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

Can I use more than 1,000 cells as input for direct cDNA synthesis using SMARTer Ultra Low kits?

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

What media have been tested for compatibility with direct cDNA synthesis from intact mammalian cells?

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)
8.00 g NaCl
1.44 g Na2HPO4 (anhydrous)
Add dH2O 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. No. 37515)
  • 0.1 ml of DMEM/F-12, GlutaMAX (Invitrogen, Cat. No. 10565) + 3.6 µl of 25% BSA (Invitrogen, Cat. No. 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 this kit.

How do I lyse cells for direct cDNA synthesis?

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 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 prior to use.

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

What is the recommended volume of Reaction Buffer for various amounts of cells when using SMARTer Ultra Low kits?

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 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 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).

Can I freeze collected cells prior to cDNA synthesis?

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, or either on ice (4°C) or at room temperature if using a legacy kit.

Can I collect cells directly in Reaction Buffer?

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 the Reaction Buffer, add fresh RNase Inhibitor after thawing cells and prior to cDNA synthesis.

FAQs for SMARTer Stranded cDNA synthesis:

SMARTer Stranded pico input kit:

What is the mechanism for ZapR-mediated removal of ribosomal RNA (rRNA) sequences from the double-stranded cDNA library?

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.

What are the R-Probes?

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.

What is ZapR?

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.

What RNA size range (nt) did Clontech test 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.

What is the smallest RNA size compatible with the SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian?

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.

Is the SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian compatible with RNA from FFPE samples?

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.

What is the cumulative size of the adapters in the SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian?

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

What is the efficiency of ZapR-mediated removal of rRNA sequences from the cDNA library?

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.

Are there any ZapR-mediated off-target effects resulting in the under-representation of certain genes in the final, double-stranded cDNA library created with the SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian?

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.

Does Clontech plan to offer ZapR-mediated removal of rRNA sequences as a separate product?

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

Can I apply the ZapR-mediated removal of rRNA sequences to my double-stranded cDNA library generated using a different library prep product, my total RNA, or my total RNA partially depleted of rRNA?

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.

Will ZapR-mediated removal of rRNA sequences work for non-mammalian samples?

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.

What is the duplicate rate in the final, double-stranded cDNA library created with the SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian?

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.

Why is the duplicate rate in the cDNA library created with the SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian higher for the lower RNA inputs?

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

Does the duplicate rate depend on the number of PCR cycles used during library amplification with the SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian?

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.

SMARTer Stranded high input kit:

How is the SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian protocol affected by the number of PCR cycles used for RNA-seq library amplification?

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 over-amplification 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.

How does the SMARTer Stranded RNA-Seq Kit maintain strand information and why is this technique better than dUTP incorporation methods?

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.

Which indexes are included in SMARTer Stranded kits?

All SMARTer Stranded kits contain 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.

How do I pool SMARTer Stranded libraries for Illumina sequencing?

The 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.

How do I analyze the quality and quantity of my cDNA sample?

How do I analyze double-stranded (ds) cDNA quality?

We recommend analyzing cDNA generated with SMARTer kits using an Agilent 2100 Bioanalyzer and the High Sensitivity DNA Chip (Agilent, Cat. No. 5067-4626) prior to sequencing.

Prior to ds 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.

Proper electropherogram for the High Sensitivity DNA ladder

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.

What is the expected size distribution of double-stranded cDNA generated by SMARTer cDNA synthesis kits?

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.

What is the expected double-stranded (ds) cDNA yield?

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 over-amplification of cDNA. See Cycling Guidelines Based on Amount of Starting Material in the user manual for your particular SMARTer Ultra Low kit.

cDNA generated with a SMARTer Ultra Low kit from various RNA amounts

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 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.

How do I determine the double-stranded (ds) cDNA yield?

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).
Determining the yield of cDNA from SMARTer Ultra Low kits Determining the yield of cDNA from SMARTer Stranded kit Determining the yield of cDNA from SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian Determining the yield of cDNA from SMARTer Universal kit

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 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 poly(A)-enriched Human Brain Total RNA (Panel B), the SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian with 1 µg Control Mouse Liver Total RNA (Panel C), or the SMARTer Universal Low Input RNA Kit for Sequencing with 2 ng chemically fragmented Human Brain Total RNA (Panel D).

What can I do if I have low cDNA yield?

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 than too many.

Low cDNA yield from a SMARTer Ultra Low kit

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.

Can I analyze unpurified double-stranded (ds) cDNA for PCR cycle optimization?

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 an 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.

Determining cDNA yield of unpurified, PCR amplification products

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.

How should sequencing libraries be prepared?

SMARTer Ultra Low kits:

What method should I use to prepare cDNA generated with SMARTer Ultra Low kits for sequencing?

We recommend two preparation methods for Illumina sequencing platforms:

  • Covaris shearing followed by the Low Input Library Prep Kit (Cat. No. 634947). This kit is compatible with 50 pg–20 ng of fragmented, double-stranded DNA (100–600 bp), allows multiplexing, and has been validated for downstream Illumina sequencing platforms.
  • The Nextera® XT DNA Sample Preparation Kit (Illumina, Cat. No. 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 (Life Technologies, Cat. No. 4471269) and an Ion Xpress Barcode Adapter kit (Life Technologies, several Cat. Nos.). This method is compatible with 1–10 ng 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.

What is the expected size range of Covaris-sheared double-stranded (ds) cDNA?

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.
Covaris-sheared cDNA prior to library prep

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

What type of Covaris machine did you use to optimize the shearing parameters?

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.

How should I set up the Peak Incident Power (W) for the Covaris S220 system?

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

What are the advantages of the Low Input Library Prep Kits?

The Low Input Library Prep Kits (Cat. Nos. 634947, 634899, and 634900) have the following advanced features:

  • A single tube, three step protocol eliminates intermediate purification steps and takes just 2 hours to complete.
  • Improved DNA end repair ensures highly efficient adapter ligation.
  • Reduced adapter background is ensured by decreased adapter-adapter ligation and removal of unused adapters after ligation.

What is the expected size range of the Covaris-sheared cDNA after library preparation?

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.

Library prep of Covaris-sheared cDNA ready for sequencing

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

How do I pool cDNA libraries generated with the Low Input Library Prep Kits for Illumina sequencing?

Follow the recommendations from Illumina for library pooling.

Can I use more than 150 pg of double-stranded (ds) cDNA for the Nextera XT DNA Sample Preparation Kit?

In our hands, using 100–150 pg of input cDNA with the Nextera XT DNA Sample Preparation Kit generates DNA fragments with 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.

Comparison of libraries generated from different cDNA input amounts

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.

Do I have to scale down the Nextera XT DNA Sample Preparation Kit protocol when using 100–150 pg of double-stranded (ds) cDNA?

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.

What is the expected size range of fragmented, double-stranded (ds) cDNA after library preparation with Nextera kits?

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.

Variability in cDNA generated from different RNA sources

Example Bioanalyzer electropherograms of cDNA libraries prepared for Illumina sequencing from 1 ng Mouse Brain Total RNA (Panel A) or 1 ng 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 Clontech. 5 ng ds cDNA and 1 µl 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.

SMARTer Stranded kits:

Illumina indexes and adapters are integrated into cDNA amplification in the SMARTer Stranded RNA-Seq Kit (including the HT version) and the SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian. No additional library preparation is needed. (see “Which indexes are included in SMARTer Stranded kits?”). Not all indexes can be pooled together, consult the Illumina literature (such as the “TruSeq DNA Sample Preparation Guide”) for appropriate pooling guidelines. When in doubt about compatibility, compare the index sequences provided in the user manuals with Illumina adapter sequences.

SMARTer Universal Low Input RNA Kit for Sequencing:

We recommend using the SMARTer Stranded RNA-Seq Kit (Cat. No. 634836) if you intend to use Illumina sequencing platforms. If you are using the SMARTer Universal kit in conjunction with Illumina sequencing, we recommend the Low Input Library Prep Kit (Cat. No. 634947). This kit generates libraries from 50 pg–20 ng of fragmented, double-stranded DNA (100–600 bp), allows multiplexing, and has been validated for downstream Illumina sequencing platforms.

For Ion Torrent sequencing, we recommend using the Ion Xpress Plus Fragment Library Preparation Kit (Life Technologies, Cat. No. 4471269) and an Ion Xpress Barcode Adapter kit (Life Technologies, several Cat. Nos.) with 1–10 ng cDNA input.

What SMARTer cDNA synthesis kits are compatible with Ion Torrent sequencing platforms?

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.

Why should I quantify my libraries prior to sequencing?

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.

Why is quantification of NGS libraries by qPCR better than using other methods?

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.

Why do library concentrations obtained with a qPCR-based method differ from those obtained by other methods?

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.

Additional tips and tricks:

Why should I perform a positive control cDNA synthesis reaction?

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.
Positive control cDNA synthesis reaction using a SMARTer Ultra Low kit

An example of an electropherogram of positive control cDNA synthesized from 100 pg Control Total RNA generated by a SMARTer Ultra Low kit using 15 cycles of PCR. The cDNA spans the expected 400–9,000 bp with a peak at approximately 2,000 bp.

Why do I have to perform a negative control during SMARTer cDNA synthesis?

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.

Negative control cDNA synthesis reaction using a SMARTer Ultra Low kit

An example electropherogram of a negative control (no input RNA) using a SMARTer Ultra Low kit with 18 cycles of PCR. No contamination is observed.

Why do I have to use different magnetic devices for SPRI bead purification of cDNA?

The two different 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.

  • MagnaBot II Magnetic Separation Device (Promega, Part No. V8351) is recommended for nuclease-free thin-wall PCR tubes (0.2 ml; USA Scientific, Cat. No. 1402-4700).
  • Magnetic Stand-96 (Life Technologies, Part No. AM10027) is recommended for 96-well V-bottom plate (500 µl; VWR, Cat. No. 47743-996).

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 distinct 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.

You may make your own magnetic separator using rare earth magnets. Click here to find out how.

How can I ensure efficient cDNA purification using SPRI beads?

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.

Can I substitute alternative products for any of the recommended Additional Materials?

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. No.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 has also been validated for use with LoBind tubes (Eppendorf Cat. No. 022431021).

  • 96-well V-bottom plates (500 µl; VWR Cat. No. 47743-996) recommended for some kits, enable a more efficient separation of SPRI beads from the supernatant when using large volumes of wash buffers.

What are the most common artifacts of cDNA synthesis with SMARTer kits?

All SMARTer kits:

  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. Improper separation of magnetic beads using a SMARTer Ultra Low kit

    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 is usually indicates contamination. A common source of contamination is the SPRI beads, which may adsorb air pollutants (e.g. pollen). cDNA generated with a SMARTer Ultra Low kit showing pollen contamination

    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.

SMARTer Ultra Low kits:

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.

Poor-quality cDNA generated with a SMARTer Ultra Low kit from degraded RNA

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.

SMARTer Stranded kits:

All Stranded kits:

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

SMARTer Universal kits:

Few reads from the sequencing run, or few clusters passing filter. SMARTer Universal libraries can have a lower than average filter pass rate due to very low complexity for the first two cycles followed by low complexity for the next five cycles, before 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.

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