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  • ‹ Back to DNA-seq
  • High-resolution CNV detection using PicoPLEX Gold DNA-Seq
  • Next-gen WGA method for CNV and SNV detection from single cells
  • Low-volume DNA shearing for ThruPLEX library prep
  • Low-input whole-exome sequencing
  • Cell-free nucleic acid sequencing
  • DNA-seq from FFPE samples
  • ThruPLEX HV data sheet
  • Improvements to ThruPLEX HV
  • ThruPLEX HV outperforms NEBNext Ultra II
  • Comparing ThruPLEX HV PLUS to Kapa and NEBNext
  • Low cell number ChIP-seq using ThruPLEX DNA-Seq
  • Accurate detection of low-frequency variants using molecular tags
Home › Learning centers › Next-generation sequencing › Technical notes › DNA-seq › Cell-free nucleic acid sequencing

Technical notes

  • DNA-seq
    • High-resolution CNV detection using PicoPLEX Gold DNA-Seq
    • Next-gen WGA method for CNV and SNV detection from single cells
    • Low-volume DNA shearing for ThruPLEX library prep
    • Low-input whole-exome sequencing
    • Cell-free nucleic acid sequencing
    • DNA-seq from FFPE samples
    • ThruPLEX HV data sheet
    • Improvements to ThruPLEX HV
    • ThruPLEX HV outperforms NEBNext Ultra II
    • Comparing ThruPLEX HV PLUS to Kapa and NEBNext
    • Low cell number ChIP-seq using ThruPLEX DNA-Seq
    • Accurate detection of low-frequency variants using molecular tags
  • Immune Profiling
    • Efficient and sensitive profiling of human B-cell receptor repertoire
    • TCRv2 kit validated for rhesus macaque samples
    • TCR repertoire profiling from mouse samples (bulk)
    • BCR repertoire profiling from mouse samples (bulk)
    • Improved TCR repertoire profiling from human samples (bulk)
    • TCR repertoire profiling from human samples (single cells)
    • BCR repertoire profiling from human samples (bulk)
  • RNA-seq
    • All-in-one cDNA synthesis and library prep from single cells
    • Automation-friendly, all-in-one cDNA synthesis and library prep
    • All-in-one cDNA synthesis and library prep from ultra-low RNA inputs
    • 3' mRNA libraries from single cells (SMART-Seq v4 3' DE Kit)
    • Full-length mRNA-seq for target capture
    • Stranded libraries from single cells
    • Stranded libraries from picogram-input total RNA (v3)
    • Stranded libraries from 100 pg-100 ng total RNA
    • Stranded libraries from 100 ng - 1 ug total RNA
    • Stranded libraries from FFPE inputs (v2)
    • Nonstranded libraries from FFPE inputs
    • Singular and Takara Bio library prep
  • Epigenetic sequencing
    • ChIP-seq libraries for transcription factor analysis
    • ChIP-seq libraries from ssDNA
    • Full-length small RNA libraries
    • Methylated DNA-seq with MBD2
  • Reproductive health technologies
    • Embgenix PGT-A (CE-IVD & RUO)
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Cell-free nucleic acid sequencing

Shorter time to better results from cell-free nucleic acids

Speed up the development of your liquid biopsy assays

"Your ThruPLEX Plasma-seq kit is the easiest to follow and has the most streamlined protocol (importantly with the fewest clean-up steps). We successfully made libraries from 1 ng input in this trial."
—Dr. Charlie Massie, UNIVERSITY OF CAMBRIDGE

ThruPLEX Plasma-Seq kit for cell-free DNA (cfDNA)

When it comes to assay development, a streamlined protocol means faster results, a need for less starting material, and less hands-on time—leading to lower variability between samples, runs, and operators, and therefore more reproducible and reliable results.

The ThruPLEX Plasma-Seq kit was developed and optimized for cell-free DNA. Its innovative chemistry offers many advantages:

  • Single-tube workflow: no transfers or purification steps required
  • Ultra-fast protocol: three steps; two hours to complete
  • Optimized reagents: no titration of adapter concentration
  • Superior performance: high-complexity NGS libraries and reproducible results

ThruPLEX Plasma-seq offers superior sensitivity and reproducibility

Figure 1. Fewer steps and pipetting operations translate to higher sensitivity and reproducibility. The ThruPLEX Plasma-Seq kit provided the most reproducible and unbiased GC coverage across the human genome, showing minimal variability across the nine plasma samples tested. Libraries were prepared from cell-free DNA isolated from an equivalent of 1 ml of plasma sample and sequenced on an Illumina NextSeq® 500 instrument. Four separate plasma samples were used to construct the NEBNext Ultra libraries.

ThruPLEX technology's high performance has enabled scientists to address challenges in obtaining insights from cell-free DNA samples, advancing the frontier of liquid biopsies to demonstrate clinical utility. Kitzman et al. prepared NGS libraries from cfDNA extracted from maternal plasma samples using a ThruPLEX Plasma-seq kit to demonstrate, for the first time, the non-invasive determination of a fetal genome sequence. Murtaza et al. used a ThruPLEX Plasma-seq kit to perform whole-exome analysis on ctDNA to monitor patients' tumor evolution before and after treatment, demonstrating the first use of NGS to non-invasively identify mutations by sequencing the cfDNA from patients. 

Recently, researchers from the Medical College of Wisconsin and the Mayo Clinic used ThruPLEX technology to identify cfDNA biomarkers for prostate, lung, and colon cancer. Watch the webinar (located below).


SMARTer Stranded Total RNA-Seq Kit v2 for cell-free RNA (cfRNA)

For sensitive detection of transcripts from Illumina-ready stranded NGS libraries generated from cell-free RNA, we offer the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Pico v2). This kit features a streamlined six-hour workflow that generates high-quality, stranded NGS libraries from 250 pg to 10 ng of purified total RNA isolated from cell-free RNA derived from plasma and other biofluids. The data shown below demonstrates the sensitivity of the kit in detecting transcripts from as little as 150 pg of input, in comparison to the ideal recommended input amount of 1 ng.

Read our tech note to learn more.

Comparison of Pico v1 versus Pico v2 kits

Figure 2. Sensitive detection of transcripts at picogram inputs of cell-free RNA with the Pico v2 kit. While the ideal input of cfRNA for the SMARTer Stranded Total RNA-seq Kit starts at 1 ng, the kit is sensitive enough to generate high-quality NGS libraries from smaller amounts of cfRNA. The data here shows the performance metrics generated using the Pico v1 kit compared to the Pico v2 kit using input amounts of 150 pg and 1 ng. The Pico v2 kit provided improved performance, yielding a low percentage of ribosomal and mitochondrial reads and a high number of genes detected.


References

Kitzman, J.O. et al. Noninvasive Whole-Genome Sequencing of a Human Fetus. Sci. Transl. Med. 4, 137–176 (2012).

Murtaza, M. et al. Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA. Nature 497, 108–112 (2013).


Mutational and copy number analysis of cell-free DNAs in human cancers

In this webinar, Dr. Liang Wang (Professor of Pathology and Molecular Genetics, Medical College of Wisconsin) shares insights from his recent work using ThruPLEX technology for next-generation sequencing of cell-free DNA to identify biomarkers for prostate, lung, and colon cancers.


Related products

Cat. # Product Size License Quantity Details
R400679 ThruPLEX® Plasma-Seq Kit 24 Rxns USD $757.00

License Statement

ID Number  
326 This product is protected by U.S. Patents 7,803,550; 8,399,199; 8,728,737, 9,598,727, 10,196,686, 10,208,337, 11,072,823 and corresponding foreign patents. Additional patents are pending. For further license information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com.
M54 This product is covered by the claims of U.S. Patent Nos. 7,704,713 and its foreign counterparts. 

The ThruPLEX Plasma-Seq Kit builds on the innovative ThruPLEX chemistry to generate high-complexity DNA libraries from cell-free DNA isolated from plasma. Single index, dual index, and unique dual index kits are available and must be purchased separately. This product contains reagents for 24 reactions.

Notice to purchaser

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.

Documents Components Image Data

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R400679: ThruPLEX Plasma-Seq Kit

R400679: ThruPLEX Plasma-Seq Kit
R400680 ThruPLEX® Plasma-Seq Kit 48 Rxns USD $1432.00

License Statement

ID Number  
326 This product is protected by U.S. Patents 7,803,550; 8,399,199; 8,728,737, 9,598,727, 10,196,686, 10,208,337, 11,072,823 and corresponding foreign patents. Additional patents are pending. For further license information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com.
M54 This product is covered by the claims of U.S. Patent Nos. 7,704,713 and its foreign counterparts. 

The ThruPLEX Plasma-Seq Kit builds on the innovative ThruPLEX chemistry to generate high-complexity DNA libraries from cell-free DNA isolated from plasma. Single index, dual index, and unique dual index kits are available and must be purchased separately. This product contains reagents for 48 reactions.

Notice to purchaser

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.

Documents Components Image Data

Back

R400680: ThruPLEX Plasma-Seq Kit

R400680: ThruPLEX Plasma-Seq Kit
R400681 ThruPLEX® Plasma-Seq Kit 96 Rxns USD $2484.00

License Statement

ID Number  
M54 This product is covered by the claims of U.S. Patent Nos. 7,704,713 and its foreign counterparts. 
326 This product is protected by U.S. Patents 7,803,550; 8,399,199; 8,728,737, 9,598,727, 10,196,686, 10,208,337, 11,072,823 and corresponding foreign patents. Additional patents are pending. For further license information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com.

The ThruPLEX Plasma-Seq Kit builds on the innovative ThruPLEX chemistry to generate high-complexity DNA libraries from cell-free DNA isolated from plasma. Single index, dual index, and unique dual index kits are available and must be purchased separately. This product contains reagents for 96 reactions.

Notice to purchaser

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.

Documents Components Image Data

Back

R400681: ThruPLEX Plasma-Seq Kit

R400681: ThruPLEX Plasma-Seq Kit
R400682 ThruPLEX® Plasma-Seq Kit 480 Rxns USD $11639.00

License Statement

ID Number  
M54 This product is covered by the claims of U.S. Patent Nos. 7,704,713 and its foreign counterparts. 
326 This product is protected by U.S. Patents 7,803,550; 8,399,199; 8,728,737, 9,598,727, 10,196,686, 10,208,337, 11,072,823 and corresponding foreign patents. Additional patents are pending. For further license information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com.

The ThruPLEX Plasma-Seq Kit builds on the innovative ThruPLEX chemistry to generate high-complexity DNA libraries from cell-free DNA isolated from plasma. Single index, dual index, and unique dual index kits are available and must be purchased separately. This product is composed of five 96-reaction kits (Cat. # R400681), 480 reactions total, of ThruPLEX Plasma-Seq reagents.

Notice to purchaser

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.

Documents Components


Related products (visit takarabio.com/picov2 for additional configurations)

Cat. # Product Size License Quantity Details
634411 SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian 12 Rxns USD $1098.00

License Statement

ID Number  
425 LIMITED USE LABEL LICENSE: RESEARCH USE ONLY Notice to Purchaser: This product is the subject to a license granted to Takara Bio USA, Inc. and its Affiliates from Caribou Biosciences, Inc., and this product is transferred to the end-user purchaser (“Purchaser”) subject to a “Limited Use Label License” conveying to the Purchaser a limited, nontransferable right to use the product, solely as provided to Purchaser, together with (i) progeny or derivatives of the product generated by the Purchaser (including but not limited to cells), and (ii) biological material extracted or derived from the product or its corresponding progeny or derivatives (including but not limited to cells) (collectively, the product, and (i) and (ii) are referred to as “Material”) only to perform internal research for the sole benefit of the Purchaser. The Purchaser cannot sell or otherwise transfer Material to a third party or otherwise use the Material for any Excluded Use. “Excluded Use” means any and all: (a) commercial activity including, but not limited to, any use in manufacturing (including but not limited to cell line development for purposes of bioproduction), product testing, or quality control; (b) preclinical or clinical testing or other activity directed toward the submission of data to the U.S. Food and Drug Administration, or any other regulatory agency in any country or jurisdiction where the active agent in such studies comprises the Material; (c) use to provide a service, information, or data to a third party with the sole exception of using the Material to conduct in vitro sample preparation, i.e., selectively depleting target cDNAs from a sample either by cleaving or selectively separating such target cDNAs from the sample through the use of the Materials; (d) use for human or animal therapeutic, diagnostic, or prophylactic purposes or as a product for therapeutics, diagnostics, or prophylaxis; (e) activity in an agricultural field trial or any activity directed toward the submission of data to the U.S. Department of Agriculture or any other agriculture regulatory agency; (f) high throughput screening drug discovery purposes (i.e., the screening of more than 10,000 experiments per day) as well as scale-up production activities for commercialization; (g) modification of human germline, including editing of human embryo genomes (with the sole exception of editing human embryonic stem (ES) cell lines for research purposes) or reproductive cells; (h) self-editing; and/or (i) stimulation of biased inheritance of a particular gene or trait or set of genes or traits (“gene drive”). It is the Purchaser’s responsibility to use the Material in accordance with all applicable laws and regulations. For information on obtaining additional rights, including commercial rights, please contact licensing@cariboubio.com or Caribou Biosciences, Inc., 2929 7th Street, Suite 105, Berkeley, CA 94710 USA, Attn: Licensing
395 This product is protected by U.S. Patent No. 10150985 and corresponding foreign patents. Additional patents are pending. For further license information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com.

The SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian is used to generate strand-specific RNA-seq libraries for Illumina sequencing from 250 pg–10 ng inputs of purified total RNA. This kit incorporates Takara Bio’s proprietary SMART (Switching Mechanism at the 5’ end of RNA Template) technology and includes refinements to the SMARTer method for stranded RNA-seq that simplify the library preparation workflow and improve sequencing performance. This method was developed to work with either high- or low-quality total RNA, does not require additional rRNA removal methods or kits, and produces sequencing libraries that retain strand-of-origin information. The integrated removal of cDNAs derived from rRNA—typically present in high abundance following cDNA synthesis from total RNA inputs—makes the workflow extremely sensitive, yielding data that is highly reproducible with low mapping to rRNA. The new library design featured in the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian improves sequencing performance compared to the original SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian, particularly for NextSeq® and MiniSeq™ instruments carrying the two-channel SBS technology. This kit includes the Indexing Primer Set HT for Illumina v2; for your convenience, we also offer the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian Components (Cat. #s 634418 and 634419) without indexing primers.

Notice to purchaser

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.

Documents Components Image Data

Back

Schematic of sequencing libraries generated with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

Schematic of sequencing libraries generated with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

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.

Back

Improved sequencing performance with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

Improved sequencing performance with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

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.

Back

Workflow for SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

Workflow for SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

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.

Back

Improved sequencing metrics with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

Improved sequencing metrics with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

Improved sensitivity and reproducibility with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian. Sequencing libraries were generated from 1 ng and 10 ng inputs of total RNA extracted from human lung FFPE tissue using both the Pico v1 and Pico v2 kits, and sequenced on a NextSeq 500 instrument. Panel A. Sequencing metrics for libraries generated from 1 ng or 10 ng inputs using each kit. For both input amounts, the Pico v2 kit resulted in greater library yields, a lower proportion of reads mapping to rRNA and mtRNA, and a lower duplicate rate. For the 1 ng input, sequencing data from the Pico v2 library also identified thousands more transcripts than sequencing data from the Pico v1 library, indicating a higher sensitivity for Pico v2. Panel B. Comparison of transcript expression levels across input amounts. Higher reproducibility was observed between 1 ng and 10 ng inputs for data generated with the Pico v2 kit vs. data generated using the Pico v1 kit. FPKM values are shown on a Log10 scale. Transcripts represented in only one library can be seen along the X- and Y-axes of the scatter plots.

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SeqAmp CB PCR buffer improves bead-pellet formation

SeqAmp CB PCR buffer improves bead-pellet formation

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 buffer (right) are tighter than those formed in the original PCR buffer (left). Tighter bead pellets tend to dry more evenly and are easier to resuspend than pellets that are broader and more diffuse.

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634411: SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

634411: SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian
634412 SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian 48 Rxns USD $3659.00

License Statement

ID Number  
425 LIMITED USE LABEL LICENSE: RESEARCH USE ONLY Notice to Purchaser: This product is the subject to a license granted to Takara Bio USA, Inc. and its Affiliates from Caribou Biosciences, Inc., and this product is transferred to the end-user purchaser (“Purchaser”) subject to a “Limited Use Label License” conveying to the Purchaser a limited, nontransferable right to use the product, solely as provided to Purchaser, together with (i) progeny or derivatives of the product generated by the Purchaser (including but not limited to cells), and (ii) biological material extracted or derived from the product or its corresponding progeny or derivatives (including but not limited to cells) (collectively, the product, and (i) and (ii) are referred to as “Material”) only to perform internal research for the sole benefit of the Purchaser. The Purchaser cannot sell or otherwise transfer Material to a third party or otherwise use the Material for any Excluded Use. “Excluded Use” means any and all: (a) commercial activity including, but not limited to, any use in manufacturing (including but not limited to cell line development for purposes of bioproduction), product testing, or quality control; (b) preclinical or clinical testing or other activity directed toward the submission of data to the U.S. Food and Drug Administration, or any other regulatory agency in any country or jurisdiction where the active agent in such studies comprises the Material; (c) use to provide a service, information, or data to a third party with the sole exception of using the Material to conduct in vitro sample preparation, i.e., selectively depleting target cDNAs from a sample either by cleaving or selectively separating such target cDNAs from the sample through the use of the Materials; (d) use for human or animal therapeutic, diagnostic, or prophylactic purposes or as a product for therapeutics, diagnostics, or prophylaxis; (e) activity in an agricultural field trial or any activity directed toward the submission of data to the U.S. Department of Agriculture or any other agriculture regulatory agency; (f) high throughput screening drug discovery purposes (i.e., the screening of more than 10,000 experiments per day) as well as scale-up production activities for commercialization; (g) modification of human germline, including editing of human embryo genomes (with the sole exception of editing human embryonic stem (ES) cell lines for research purposes) or reproductive cells; (h) self-editing; and/or (i) stimulation of biased inheritance of a particular gene or trait or set of genes or traits (“gene drive”). It is the Purchaser’s responsibility to use the Material in accordance with all applicable laws and regulations. For information on obtaining additional rights, including commercial rights, please contact licensing@cariboubio.com or Caribou Biosciences, Inc., 2929 7th Street, Suite 105, Berkeley, CA 94710 USA, Attn: Licensing
395 This product is protected by U.S. Patent No. 10150985 and corresponding foreign patents. Additional patents are pending. For further license information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com.

The SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian is used to generate strand-specific RNA-seq libraries for Illumina sequencing from 250 pg–10 ng inputs of purified total RNA. This kit incorporates Takara Bio’s proprietary SMART (Switching Mechanism at the 5’ end of RNA Template) technology and includes refinements to the SMARTer method for stranded RNA-seq that simplify the library preparation workflow and improve sequencing performance. This method was developed to work with either high- or low-quality total RNA, does not require additional rRNA removal methods or kits, and produces sequencing libraries that retain strand-of-origin information. The integrated removal of cDNAs derived from rRNA—typically present in high abundance following cDNA synthesis from total RNA inputs—makes the workflow extremely sensitive, yielding data that is highly reproducible with low mapping to rRNA. The new library design featured in the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian improves sequencing performance compared to the original SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian, particularly for NextSeq® and MiniSeq™ instruments carrying the two-channel SBS technology. This kit includes the Indexing Primer Set HT for Illumina v2; for your convenience, we also offer the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian Components (Cat. #s 634418 and 634419) without indexing primers.

Notice to purchaser

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.

Documents Components Image Data

Back

Schematic of sequencing libraries generated with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

Schematic of sequencing libraries generated with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

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.

Back

Improved sequencing performance with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

Improved sequencing performance with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

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.

Back

Workflow for SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

Workflow for SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

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.

Back

Improved sequencing metrics with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

Improved sequencing metrics with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

Improved sensitivity and reproducibility with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian. Sequencing libraries were generated from 1 ng and 10 ng inputs of total RNA extracted from human lung FFPE tissue using both the Pico v1 and Pico v2 kits, and sequenced on a NextSeq 500 instrument. Panel A. Sequencing metrics for libraries generated from 1 ng or 10 ng inputs using each kit. For both input amounts, the Pico v2 kit resulted in greater library yields, a lower proportion of reads mapping to rRNA and mtRNA, and a lower duplicate rate. For the 1 ng input, sequencing data from the Pico v2 library also identified thousands more transcripts than sequencing data from the Pico v1 library, indicating a higher sensitivity for Pico v2. Panel B. Comparison of transcript expression levels across input amounts. Higher reproducibility was observed between 1 ng and 10 ng inputs for data generated with the Pico v2 kit vs. data generated using the Pico v1 kit. FPKM values are shown on a Log10 scale. Transcripts represented in only one library can be seen along the X- and Y-axes of the scatter plots.

Back

SeqAmp CB PCR buffer improves bead-pellet formation

SeqAmp CB PCR buffer improves bead-pellet formation

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 buffer (right) are tighter than those formed in the original PCR buffer (left). Tighter bead pellets tend to dry more evenly and are easier to resuspend than pellets that are broader and more diffuse.

Back

634412: SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

634412: SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian
634413 SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian 96 Rxns USD $4811.00

License Statement

ID Number  
425 LIMITED USE LABEL LICENSE: RESEARCH USE ONLY Notice to Purchaser: This product is the subject to a license granted to Takara Bio USA, Inc. and its Affiliates from Caribou Biosciences, Inc., and this product is transferred to the end-user purchaser (“Purchaser”) subject to a “Limited Use Label License” conveying to the Purchaser a limited, nontransferable right to use the product, solely as provided to Purchaser, together with (i) progeny or derivatives of the product generated by the Purchaser (including but not limited to cells), and (ii) biological material extracted or derived from the product or its corresponding progeny or derivatives (including but not limited to cells) (collectively, the product, and (i) and (ii) are referred to as “Material”) only to perform internal research for the sole benefit of the Purchaser. The Purchaser cannot sell or otherwise transfer Material to a third party or otherwise use the Material for any Excluded Use. “Excluded Use” means any and all: (a) commercial activity including, but not limited to, any use in manufacturing (including but not limited to cell line development for purposes of bioproduction), product testing, or quality control; (b) preclinical or clinical testing or other activity directed toward the submission of data to the U.S. Food and Drug Administration, or any other regulatory agency in any country or jurisdiction where the active agent in such studies comprises the Material; (c) use to provide a service, information, or data to a third party with the sole exception of using the Material to conduct in vitro sample preparation, i.e., selectively depleting target cDNAs from a sample either by cleaving or selectively separating such target cDNAs from the sample through the use of the Materials; (d) use for human or animal therapeutic, diagnostic, or prophylactic purposes or as a product for therapeutics, diagnostics, or prophylaxis; (e) activity in an agricultural field trial or any activity directed toward the submission of data to the U.S. Department of Agriculture or any other agriculture regulatory agency; (f) high throughput screening drug discovery purposes (i.e., the screening of more than 10,000 experiments per day) as well as scale-up production activities for commercialization; (g) modification of human germline, including editing of human embryo genomes (with the sole exception of editing human embryonic stem (ES) cell lines for research purposes) or reproductive cells; (h) self-editing; and/or (i) stimulation of biased inheritance of a particular gene or trait or set of genes or traits (“gene drive”). It is the Purchaser’s responsibility to use the Material in accordance with all applicable laws and regulations. For information on obtaining additional rights, including commercial rights, please contact licensing@cariboubio.com or Caribou Biosciences, Inc., 2929 7th Street, Suite 105, Berkeley, CA 94710 USA, Attn: Licensing
395 This product is protected by U.S. Patent No. 10150985 and corresponding foreign patents. Additional patents are pending. For further license information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com.

The SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian is used to generate strand-specific RNA-seq libraries for Illumina sequencing from 250 pg–10 ng inputs of purified total RNA. This kit incorporates Takara Bio’s proprietary SMART (Switching Mechanism at the 5’ end of RNA Template) technology and includes refinements to the SMARTer method for stranded RNA-seq that simplify the library preparation workflow and improve sequencing performance. This method was developed to work with either high- or low-quality total RNA, does not require additional rRNA removal methods or kits, and produces sequencing libraries that retain strand-of-origin information. The integrated removal of cDNAs derived from rRNA—typically present in high abundance following cDNA synthesis from total RNA inputs—makes the workflow extremely sensitive, yielding data that is highly reproducible with low mapping to rRNA. The new library design featured in the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian improves sequencing performance compared to the original SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian, particularly for NextSeq® and MiniSeq™ instruments carrying the two-channel SBS technology. This kit includes the Indexing Primer Set HT for Illumina v2; for your convenience, we also offer the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian Components (Cat. #s 634418 and 634419) without indexing primers.

Notice to purchaser

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.

Documents Components Image Data

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Schematic of sequencing libraries generated with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

Schematic of sequencing libraries generated with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

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.

Back

Improved sequencing performance with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

Improved sequencing performance with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

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.

Back

Workflow for SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

Workflow for SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

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.

Back

Improved sequencing metrics with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

Improved sequencing metrics with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

Improved sensitivity and reproducibility with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian. Sequencing libraries were generated from 1 ng and 10 ng inputs of total RNA extracted from human lung FFPE tissue using both the Pico v1 and Pico v2 kits, and sequenced on a NextSeq 500 instrument. Panel A. Sequencing metrics for libraries generated from 1 ng or 10 ng inputs using each kit. For both input amounts, the Pico v2 kit resulted in greater library yields, a lower proportion of reads mapping to rRNA and mtRNA, and a lower duplicate rate. For the 1 ng input, sequencing data from the Pico v2 library also identified thousands more transcripts than sequencing data from the Pico v1 library, indicating a higher sensitivity for Pico v2. Panel B. Comparison of transcript expression levels across input amounts. Higher reproducibility was observed between 1 ng and 10 ng inputs for data generated with the Pico v2 kit vs. data generated using the Pico v1 kit. FPKM values are shown on a Log10 scale. Transcripts represented in only one library can be seen along the X- and Y-axes of the scatter plots.

Back

SeqAmp CB PCR buffer improves bead-pellet formation

SeqAmp CB PCR buffer improves bead-pellet formation

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 buffer (right) are tighter than those formed in the original PCR buffer (left). Tighter bead pellets tend to dry more evenly and are easier to resuspend than pellets that are broader and more diffuse.

Back

634413: SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

634413: SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian
634414 SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian 192 Rxns Inquire for Quotation

License Statement

ID Number  
425 LIMITED USE LABEL LICENSE: RESEARCH USE ONLY Notice to Purchaser: This product is the subject to a license granted to Takara Bio USA, Inc. and its Affiliates from Caribou Biosciences, Inc., and this product is transferred to the end-user purchaser (“Purchaser”) subject to a “Limited Use Label License” conveying to the Purchaser a limited, nontransferable right to use the product, solely as provided to Purchaser, together with (i) progeny or derivatives of the product generated by the Purchaser (including but not limited to cells), and (ii) biological material extracted or derived from the product or its corresponding progeny or derivatives (including but not limited to cells) (collectively, the product, and (i) and (ii) are referred to as “Material”) only to perform internal research for the sole benefit of the Purchaser. The Purchaser cannot sell or otherwise transfer Material to a third party or otherwise use the Material for any Excluded Use. “Excluded Use” means any and all: (a) commercial activity including, but not limited to, any use in manufacturing (including but not limited to cell line development for purposes of bioproduction), product testing, or quality control; (b) preclinical or clinical testing or other activity directed toward the submission of data to the U.S. Food and Drug Administration, or any other regulatory agency in any country or jurisdiction where the active agent in such studies comprises the Material; (c) use to provide a service, information, or data to a third party with the sole exception of using the Material to conduct in vitro sample preparation, i.e., selectively depleting target cDNAs from a sample either by cleaving or selectively separating such target cDNAs from the sample through the use of the Materials; (d) use for human or animal therapeutic, diagnostic, or prophylactic purposes or as a product for therapeutics, diagnostics, or prophylaxis; (e) activity in an agricultural field trial or any activity directed toward the submission of data to the U.S. Department of Agriculture or any other agriculture regulatory agency; (f) high throughput screening drug discovery purposes (i.e., the screening of more than 10,000 experiments per day) as well as scale-up production activities for commercialization; (g) modification of human germline, including editing of human embryo genomes (with the sole exception of editing human embryonic stem (ES) cell lines for research purposes) or reproductive cells; (h) self-editing; and/or (i) stimulation of biased inheritance of a particular gene or trait or set of genes or traits (“gene drive”). It is the Purchaser’s responsibility to use the Material in accordance with all applicable laws and regulations. For information on obtaining additional rights, including commercial rights, please contact licensing@cariboubio.com or Caribou Biosciences, Inc., 2929 7th Street, Suite 105, Berkeley, CA 94710 USA, Attn: Licensing
395 This product is protected by U.S. Patent No. 10150985 and corresponding foreign patents. Additional patents are pending. For further license information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com.
*

The SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian is used to generate strand-specific RNA-seq libraries for Illumina sequencing from 250 pg–10 ng inputs of purified total RNA. This kit incorporates Takara Bio’s proprietary SMART (Switching Mechanism at the 5’ end of RNA Template) technology and includes refinements to the SMARTer method for stranded RNA-seq that simplify the library preparation workflow and improve sequencing performance. This method was developed to work with either high- or low-quality total RNA, does not require additional rRNA removal methods or kits, and produces sequencing libraries that retain strand-of-origin information. The integrated removal of cDNAs derived from rRNA—typically present in high abundance following cDNA synthesis from total RNA inputs—makes the workflow extremely sensitive, yielding data that is highly reproducible with low mapping to rRNA. The new library design featured in the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian improves sequencing performance compared to the original SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian, particularly for NextSeq® and MiniSeq™ instruments carrying the two-channel SBS technology. This kit includes the Indexing Primer Set HT for Illumina v2; for your convenience, we also offer the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian Components (Cat. #s 634418 and 634419) without indexing primers.

Notice to purchaser

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.

Documents Components Image Data

Back

Schematic of sequencing libraries generated with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

Schematic of sequencing libraries generated with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

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.

Back

Improved sequencing performance with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

Improved sequencing performance with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

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.

Back

Workflow for SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

Workflow for SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

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.

Back

Improved sequencing metrics with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

Improved sequencing metrics with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

Improved sensitivity and reproducibility with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian. Sequencing libraries were generated from 1 ng and 10 ng inputs of total RNA extracted from human lung FFPE tissue using both the Pico v1 and Pico v2 kits, and sequenced on a NextSeq 500 instrument. Panel A. Sequencing metrics for libraries generated from 1 ng or 10 ng inputs using each kit. For both input amounts, the Pico v2 kit resulted in greater library yields, a lower proportion of reads mapping to rRNA and mtRNA, and a lower duplicate rate. For the 1 ng input, sequencing data from the Pico v2 library also identified thousands more transcripts than sequencing data from the Pico v1 library, indicating a higher sensitivity for Pico v2. Panel B. Comparison of transcript expression levels across input amounts. Higher reproducibility was observed between 1 ng and 10 ng inputs for data generated with the Pico v2 kit vs. data generated using the Pico v1 kit. FPKM values are shown on a Log10 scale. Transcripts represented in only one library can be seen along the X- and Y-axes of the scatter plots.

Back

SeqAmp CB PCR buffer improves bead-pellet formation

SeqAmp CB PCR buffer improves bead-pellet formation

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 buffer (right) are tighter than those formed in the original PCR buffer (left). Tighter bead pellets tend to dry more evenly and are easier to resuspend than pellets that are broader and more diffuse.

Back

634414: SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

634414: SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

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Takara Bio USA, Inc. provides kits, reagents, instruments, and services that help researchers explore questions about gene discovery, regulation, and function. As a member of the Takara Bio Group, Takara Bio USA is part of a company that holds a leadership position in the global market and is committed to improving the human condition through biotechnology. Our mission is to develop high-quality innovative tools and services to accelerate discovery.

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That's GOOD Science!

What does it take to generate good science? Careful planning, dedicated researchers, and the right tools. At Takara Bio, we thoughtfully develop best-in-class products to tackle your most challenging research problems, and have an expert team of technical support professionals to help you along the way, all at superior value.

Explore what makes good science possible

Mutational and copy number analysis of cell-free DNAs in human cancers

In this webinar, Dr. Liang Wang (Professor of Pathology and Molecular Genetics, Medical College of Wisconsin) shares insights from his recent work using ThruPLEX technology for next-generation sequencing of cell-free DNA to identify biomarkers for prostate, lung, and colon cancers.

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Takara Bio USA, Inc. provides kits, reagents, instruments, and services that help researchers explore questions about gene discovery, regulation, and function. As a member of the Takara Bio Group, Takara Bio USA is part of a company that holds a leadership position in the global market and is committed to improving the human condition through biotechnology. Our mission is to develop high-quality innovative tools and services to accelerate discovery.

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES (EXCEPT AS SPECIFICALLY NOTED).

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