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SMART-Seq v4 Reagent Kit for the Apollo System Apollo SMART-Seq v4 product page
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Tech Note

Automating sensitive SMART-Seq v4 chemistry for reliable, high-throughput results on the Apollo system

Introduction Results Conclusion Methods

Introduction  

As researchers delve deeper into the human genome, demand for highly sensitive and reproducible RNA-seq is growing. Next-generation sequencing (NGS) applications offer diverse and detailed analysis for these studies, but manual library preparation is repetitive, tedious, and prone to human error and variability. In contrast to manual preparation methods, the Apollo system brings the benefits of high-throughput automation to the equation. By combining the industry-leading sensitivity of SMART-Seq v4 (SSv4) ultra-low-input cDNA synthesis with the consistency of the Apollo instrument, we developed an automated workflow for ultra-low-input and single-cell RNA-seq, yielding comparable, if not better, results for cDNA synthesis versus manual protocols, while also decreasing hands-on time and variability.

Results  

To analyze the use of SSv4 chemistry on the automated system, cDNA amplification for library preparation was performed on three different Apollo instruments with 48 samples per run (40 with Control Total RNA and eight no-RNA controls). For each run, four bench controls (three with Control Total RNA and one no-RNA control) were performed manually using the same master mixes and sharing the same thermal cycler as the automated samples.

Similar profiles and yields for automated and manual cDNA amplification

Amplified cDNA was analyzed with an Agilent 2100 Bioanalyzer and Agilent's High Sensitivity DNA Kit (Figure 1). Results showed similar amplified cDNA profiles between automated replicates, demonstrating consistency between runs. These automated replicates also showed similar profiles when compared to the manually generated cDNA, indicating that the SSv4 technology performs just as well under these time-saving, high-throughput conditions as it does with manual benchtop workflows.

Apollo bioanalyzer profile

Figure 1. Bioanalyzer profiles for amplified cDNA between automated and manual protocols. Amplified cDNA profiles demonstrate consistency between automated replicates and between automated and manual workflows.

The yield was calculated for the region from 300–9,000 bp and then averaged across the three Apollo runs, with 40 samples each. The three sets of manual controls with three samples each were subjected to the same calculations. Overall yield was not significantly different between the two amplification methods (Table 1).

Workflow Number of samples Average yield (ng)
Overall Manual 9 5.8
Apollo 120 5.0

Table 1. Bioanalyzer quantification of automated replicates compared to manual samples.

Sequencing libraries generated using automated and manual amplification protocols show similar metrics and high correlations

A subset of the cDNAs amplified above (33 Apollo-generated samples and nine manually generated samples) were used in a manual Nextera® XT protocol for library preparation. The resulting libraries were sequenced on a NextSeq® instrument with 75-bp paired-end reads. Reads were trimmed and mapped to rRNA and the mitochondrial genome. Remaining unmapped reads were mapped to the mouse genome, producing uniquely mapped reads and percentage of reads that mapped to RefSeq annotations, including exons, introns, and intergenic regions. The number of transcripts identified in each library was determined by the number of transcripts with an FPKM ≥0.1 or FPKM ≥1. Sequencing metrics from each of these runs show similar results for libraries generated from cDNA amplified by either workflow (Table 2).

  Workflow Number of samples % rRNA Number of transcripts
(FPKM > 0.1)
Number of transcripts
(FPKM > 1)
Range of Pearson correlations
Overall Manual 9 0.9 11,609 10,201 0.94–0.97
Apollo 33 1.7 12,454 10,746 0.91–1.0

Table 2. Similar sequencing metrics between libraries generated from cDNA amplified by automated and manual protocols. FPKM = fragments per kb mapped.

Libraries generated from both types of samples showed high correlation between those from the manual workflow and those from the Apollo instrument (Figure 2), indicating that scaling up to a high-throughput protocol does not affect the capabilities of the SMART-Seq chemistry.

Apollo scatter plot

Figure 2. High correlation between libraries generated from manual and automated cDNA amplification samples. Data for each workflow represents an average of at least three technical replicates. Expression measurements are reported as FPKM.

Conclusion  

Automation of our sensitive SMART-Seq v4 technology for library preparation for ultra-low-input or single-cell RNA-seq on the Apollo system provides researchers with a walkaway solution to high-throughput cDNA synthesis and downstream NGS applications. This high-throughput system displays consistency between automated replicates and provides results that show a high similarity with those of manually generated libraries, without the tedious manual processing. Learn more about the Apollo system and how it increases reproducibility while minimizing sample loss by reading our in-tip bead separation tech note.

Methods  

Automated cDNA amplification was performed on three Apollo instruments with 48 samples in each run (40 with 10 pg of Control Total RNA as input, eight negative controls with no RNA input). For each of the three runs, four bench controls were performed manually, using the same master mixes as were made for the automated runs, and also sharing the same thermal cycler. The bench controls included three reactions with 10 pg of Control Total RNA as input and one negative control with no RNA input. cDNA amplification was performed according to the protocol in the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing User Manual, using 17 PCR cycles.

1 µl of amplified cDNA was analyzed with an Agilent 2100 Bioanalzyer and a High Sensitivity DNA Kit (Agilent, Cat. # 5067-4326). The peak was manually integrated using the Bioanalyzer for the region from 300–9,000 bp to calculate the concentration. To calculate the total cDNA output, the concentration (as defined by the Bioanalyzer software) was multiplied by 17 µl. Averages were calculated across three Apollo runs with 40 samples each, and three sets of manual controls with three samples each.

100 pg of amplified cDNA was used in a manual Nextera XT protocol. The resulting sequencing libraries were sequenced on a NextSeq instrument with paired-end 75-bp reads. Analysis was performed with CLC Bio v9.5.3. Reads were trimmed and mapped to rRNA and the mitochondrial genome. Unmapped reads were then mapped to the mouse genome (mm9), producing uniquely mapped reads and percent reads that mapped to RefSeq annotations, including exons, introns, and intergenic regions. The number of transcripts identified in each library was determined by the number of transcripts with an FPKM ≥0.1 or FPKM ≥1. Expression levels of different genes were obtained using CLC Genomics Workbench after mapping to RefSeq. Transcript expression levels were exported from CLC, and correlations between individual samples (data not shown) or groups of samples (data shown in Figure 2) were obtained using Excel.

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Cat. # Product Size License Quantity Details
640078 Apollo™ Library Prep System Each Inquire for Quotation *

The Apollo Library Prep System is an all-inclusive benchtop automated liquid handler that provides reliable, walkaway NGS library preparation. Its validated scripts and optimized, plug-and-play PrepX chemistries for DNA-, RNA-, and ChIP-seq increase reproducibility and reduce hands-on time as compared to manual library prep methods.

 

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Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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In-tip bead separation improves reproducibility of libraries.

In-tip bead separation improves reproducibility of libraries.

In-tip bead separation improves reproducibility of final libraries, regardless of deck position or time of sample processing. DNA-seq libraries were prepared from 1 µg of human brain total DNA using the SMARTer PrepX Complete ILM 32i DNA Library Kit, 96 Samples. Bioanalyzer traces of libraries produced from different deck positions and on different days with the same input sample demonstrate consistent library preparation, independent of when the sample is processed or which deck position is used for processing.

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Poly A+ RNA enrichment on the SMARTer Apollo system produces better quality and yield as compared to manual poly A+ RNA enrichment.

Poly A+ RNA enrichment on the SMARTer Apollo system produces better quality and yield as compared to manual poly A+ RNA enrichment.

Poly A+ RNA enrichment on the SMARTer Apollo system produces better quality and yield as compared to manual poly A+ RNA enrichment. Panel A. Manual poly A+ RNA enrichment was performed on 1 µg of human brain total RNA in triplicate. Bioanalyzer traces of these manual replicates show variation in yield. Panel B. Automated poly A+ RNA enrichment was performed on 1 µg of human brain total RNA and a no-template control using the SMARTer Apollo system with the SMARTer PrepX PolyA mRNA Isolation Kit, 96 Samples. Automated enrichment (Panel B, red trace) results in higher poly A+ RNA yield and diversity as compared to manual enrichment (Panel A).

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Consistent library size with a SMARTer PrepX DNA library kit

Consistent library size with a SMARTer PrepX DNA library kit

Consistent library size with a SMARTer PrepX DNA library kit. Eight technical replicate E. coli libraries were created with a SMARTer PrepX DNA library kit on the SMARTer Apollo system, demonstrating consistent post-PCR library sizes.

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In-tip bead separation maximizes reproducibility.

In-tip bead separation maximizes reproducibility.

In-tip bead separation maximizes reproducibility. Improved bead separation is facilitated by an automated magnetic arm that comes into contact with the tips. Panel A. First, the SMARTer Apollo system draws up the suspended beads (the orange liquid). Then, the magnetic bar swings down and makes contact with the tips, instantly attracting the beads to the magnet. Any remaining beads are captured by repeatedly passing the solution in the tip over the magnetic bar. Panel B. After capture, the beads can be seen as a dark mass adhering to the magnetic arm.

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640078: Apollo Library Prep System

640078: Apollo Library Prep System
640170 SMART-Seq® v4 Reagent Kit for the Apollo™ System 96 Rxns USD $5964.00

License Statement

ID Number  
275 SMART-Seq2 Technology. This product is sold under exclusive license from Ludwig Institute of Cancer Research, Ltd. and is covered by US Patent No. 10266894, Japanese Patent No. 6336080, and European Patent No. 3036336, and pending U.S. patent application and/or pending claims of foreign counterparts. For license information, please contact a Takara Bio USA, Inc. licensing representative by phone at 650.919.7320 or by e-mail at licensing@takarabio.com.

The SMART-Seq v4 Reagent Kit for the SMARTer Apollo System is designed to generate high-quality cDNA from 10 pg–10 ng of total RNA or directly from 1–1,000 cells. This kit is specifically formulated for the SMARTer Apollo system, enabling processing of up to 96 samples in a single working day with minimal hands-on time. cDNA produced with the kit can be used to construct RNA-seq libraries that are compatible with Illumina® or Ion Torrent platforms, and that provide industry-leading sensitivity and accurate representation of rare and GC-rich transcripts.

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Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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640170: SMART-Seq v4 Reagent Kit for the SMARTer Apollo System

640170: SMART-Seq v4 Reagent Kit for the SMARTer Apollo System


Apollo instrument

Automated, high-throughput NGS library preparation from DNA, RNA, or ChIP DNA.

Apollo reagents

Chemistries and protocols for DNA-seq, RNA-seq, and ChIP-seq NGS library prep.

SMART-Seq v4 for Apollo

Automated SMART-Seq v4 workflow performs like your manual workflow—but faster.

Apollo consumables

Reservoirs, tips, tubes, caps, plates, and other plastics for automated NGS library prep.

Technology overview

Get unrivaled results with simplified, automated NGS library preparation.

In-tip bead separation

Apollo system's unique library cleanup enables reliable NGS library prep.

Apollo citations

See how scientists around the world use the Apollo system.

Technical specifications

Detailed technical specifications of the Apollo instrument.

Apollo services

Available service options for the Apollo system.

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