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SMART-Seq mRNA Long Read

SMART-Seq mRNA Long Read generates high-quality full-length barcoded cDNA from ultra-low inputs for long-read sequencing on the Oxford Nanopore Technologies (ONT) instruments. The workflow is optimized for 10 pg–100 ng high integrity total RNA (RIN >8) or 1–1,000 intact cells. Up to 96 multiplexed libraries can be prepared for single-tube ONT library preparation. Enjoy easy and convenient demultiplexing using a protocol and files provided by Takara Bio for use with Dorado from ONT.

SMART-Seq mRNA Long Read enables whole-transcriptome analysis from low-input samples using long-read sequencing. The workflow uses dT priming to generate full-length cDNA from 10 pg–100 ng high integrity total RNA (RIN >8) or directly from 1–1,000 intact cells. Subsequent cDNA amplification using barcoded primers allows the preparation of up to 96 barcoded cDNA libraries for subsequent adapter ligation and library prep for long-read sequencing on the Oxford Nanopore Technologies (ONT) platform. This strategy enables multiplexing of low-input samples for ONT library preparation, reliably generating average read lengths (N50) of ~2 kb and detecting full-length transcripts over 8 kb.

The complete protocol including cDNA synthesis, barcoding, enrichment, and pooling of barcoded cDNA for ONT library prep can be completed in one and a half days. The workflow is automation and miniaturization friendly. Sequencing data generated with this kit can easily be demultiplexed using a protocol and files provided by Takara Bio for use with Dorado from ONT.

The SMART (Switching Mechanism at 5' End of RNA Template) technology powering cDNA synthesis provides an efficient way to capture full-length transcript information, enabling analysis of transcript isoforms, gene fusions, point mutations, etc. Additionally, the highly sensitive and reproducible chemistry enables the identification of higher numbers of genes relative to other methods, even from low-input samples. The kits provide high reproducibility, even gene-body coverage, and an accurate representation of GC-rich transcripts.

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Cat. #	Product	Size	Price
634376	SMART-Seq® mRNA Long Read	96 Rxns	USD $3231.00
SMART-Seq mRNA Long Read generates full-length, barcoded cDNA directly from 1–1,000 intact cells or 10 pg–100 ng of total RNA in a convenient input volume of 1–9.5 µl. The full workflow can be completed within 2 days. 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 Library preparation workflow for the SMART-Seq mRNA Long Read kit. Library preparation workflow for the SMART-Seq mRNA Long Read kit. Starting with high-integrity RNA (RIN≥8) or intact cells, first-strand cDNA synthesis is primed by the SMART-Seq LR Oligo-dT and performed by an MMLV-derived reverse transcriptase (RT). Upon reaching the 5' end of each mRNA molecule, the RT adds nontemplated nucleotides to the first-strand cDNA. The SMART-Seq LR TSO contains a sequence that is complementary to the nontemplated nucleotides added by the RT. This primer hybridizes to the first-strand cDNA. In the template switching step, the RT uses the remainder of the SMART-Seq LR TSO as a template for the incorporation of an additional sequence on the end of the first-strand cDNA. The first-strand cDNA is then barcoded by forward and reverse primers from the SMART-Seq Long Read Index Kit (96 rxns) and amplified by the first PCR; a second PCR enriches for barcoded fragments. Samples are pooled and end-prepped, and sequencing adapters are ligated using the Ligation Sequencing Kit V14 (ONT). After sequencing, samples are basecalled and demultiplexed using ONT's Guppy. Downstream data analysis was performed using Cutadapt, Minimap2, SAMtools, Bedtools, and Salmon. Back SMART-Seq mRNA LR generates barcoded cDNA with a wide range of fragment lengths. SMART-Seq mRNA LR generates barcoded cDNA with a wide range of fragment lengths. SMART-Seq mRNA LR workflow was used to create cDNA from 10 pg or 10 ng total mouse brain RNA (n = 8). cDNA size distribution was measured on a 2100 Bioanalyzer (Agilent Technologies) using an Agilent High Sensitivity DNA Kit. Panel A. Barcoded cDNA was pooled per input, and libraries were generated using the Ligation Sequencing Kit V14. Libraries were sequenced on a MinION Flow Cell (ONT) for 72 hr. Samples were basecalled and demultiplexed using ONTs' Guppy and read-length distribution was plotted using MS-Excel. Panels B & C. Read-length distributions are shown for a representative sample of 10 pg (Panel B) and 10 ng (Panel C) total mouse brain RNA. Back SMART-Seq mRNA Long Read demonstrates high sensitivity across a broad range of inputs. SMART-Seq mRNA Long Read demonstrates high sensitivity across a broad range of inputs. To evaluate the performance, cDNA and sequencing libraries were generated from 10 pg and 10 ng total mouse brain RNA (MBR) using the SMART-Seq mRNA LR workflow. After sequencing, data was basecalled and demultiplexed using ONT's Guppy, and reads per barcode were downsampled to 400,000 reads. The read distribution for 10 pg and 10 ng MBR input. Panel A. is similar, showing consistent performance across the input range (n = 8 for 10 ng; n = 7 for 10 pg). Panel B. Reads of the 10 pg MBR dataset downsampled to assess gene and transcript sensitivity. Panel C. 10 ng MBR dataset downsampled to assess gene and transcript sensitivity. While neither of the two conditions (10 pg and 10 ng) were sequenced until saturation, both highlight the high gene and transcript sensitivity across read depths. Back SMART-Seq mRNA LR is highly reproducible across replicates for a broad range of inputs. SMART-Seq mRNA LR is highly reproducible across replicates for a broad range of inputs. The SMART-Seq mRNA LR workflow was used to create cDNA from 10 ng MBR of 96 replicates. Barcoded cDNA of all 96 samples were pooled, and libraries were generated and sequenced according to the workflow. Back SMART-Seq mRNA LR yields even gene-body coverage for ultra-low inputs. SMART-Seq mRNA LR yields even gene-body coverage for ultra-low inputs. 10 pg and 10 ng MBR samples underwent the SMART-Seq mRNA LR workflow. Gene-body coverage was assessed for the average of eight replicates of 10 pg and 10 ng MBR samples. Back SMART-Seq mRNA LR is compatible with direct cell inputs. SMART-Seq mRNA LR is compatible with direct cell inputs. cDNA was generated from single K562 cells or 1,000 K562 cells using the SMART-Seq mRNA LR workflow. After sequencing, data was basecalled and demultiplexed using Guppy, and reads per barcode were downsampled to 300,000 reads. Panel A. The average gene count for each condition is shown above the corresponding bar (n = 8 for single cell and n = 2 for 1,000 cells). Panel B. Pearson correlation was calculated from the gene matrix across eight single K562 cells. Panel C. Downsampling analysis of single K562 cells shows gene and transcript count per read depth. Back SMART-Seq mRNA LR detects full-length isoforms and gene fusions. SMART-Seq mRNA LR detects full-length isoforms and gene fusions. cDNA was generated using the SMART-Seq mRNA LR workflow. Basecalling and demultiplexing were performed using ONT's G uppy, and reads were aligned using Minimap2. Panels A & B. Isoforms of Snap25 (Panel A) and Nbr1 (Panel B) detected from 10 pg MBR input are visualized in IGV. Panel C. NUP214::XKR3 gene fusions detected from single-cell input are visualized in IGV. Back SMART-Seq mRNA LR is compatible with automation and miniaturization. SMART-Seq mRNA LR is compatible with automation and miniaturization. The SMART-Seq mRNA LR workflow was used to create cDNA from 10 ng MBR of 96 replicates manually on the benchtop with the full reaction volumes or automated at 1/8-volume on a mosquito HV (SPT Labtech). Barcoded cDNA of all 96 samples per experiment was pooled, and libraries were generated and sequenced for 72 hr according to the workflow. Samples were basecalled and demultiplexed using ONT Guppy and aligned with Minimap2. Panel A. Render of the mosquito HV liquid handler. Panel B. Gene counts were assessed with Salmon. Panel C. Mapping statistics of manually prepared cDNA compared to cDNA prepared using the mosquito HV. Back SMART-Seq mRNA LR performance with mRNA reference standards SMART-Seq mRNA LR performance with mRNA reference standards. To evaluate the performance of the SMART-Seq mRNA LR workflow, cDNA was generated from SIRV-Set 4 (Lexogen) mRNA reference standards which contains both ERCC quantification controls and Long SIRV mRNA standards. 10 ng of mouse brain total RNA was prepared—with SIRV spike-ins added to account for approximately 5% of reads—using the workflow described in Figure 1. Libraries were sequenced by ONT MinION, FASTQ data was read-strand corrected using the Restrander tool, and data were aligned with minimap2. ERCC standard abundance for measured vs. theoretical concentration was plotted. Back SMART-Seq mRNA LR performance with mRNA reference standards SMART-Seq mRNA LR performance with mRNA reference standards. To evaluate the performance of the SMART-Seq mRNA LR workflow, cDNA was generated from SIRV-Set 4 (Lexogen) mRNA reference standards which contains both ERCC quantification controls and Long SIRV mRNA standards. 10 ng of mouse brain total RNA was prepared—with SIRV spike-ins added to account for approximately 5% of reads—using the workflow described in Figure 1. Libraries were sequenced by ONT MinION, FASTQ data was read-strand corrected using the Restrander tool, and data were aligned with minimap2. IGV plots of data from 1 kb, 4 kb, 6 kb, and 8 kb long transcripts from the ERCC and long SIRV isoform set. Red colored reads indicate positive strand reads, blue indicates very rare negative-strand reads, and small purple or red marks indicate small indels/variations common in ONT-sequencing. Full-length is defined as reads that cover at least 90% of expected mRNA length. Back Visualization showing completeness of fragment lengths and strand orientation accuracy of the SSmRNA LR kit Visualization showing completeness of fragment lengths and strand orientation accuracy of the SSmRNA LR kit. This experiment used Spike-In RNA Variant (SIRV) controls (600–2,492 bp), showcasing detection of full-length fragments with a high percentage of complete transcripts, and accurate identification of strand orientation across all isoforms. Panel A. Coverage analysis of complete, full-length fragments. Panel B . Strand identification across different SIRV-spike-in species visualized via SIRVsuite. Back Automation- and miniaturization-friendly SSmRNA LR workflow comparison SMART-Seq mRNA LR performance with mRNA reference standards. To evaluate the performance of the SMART-Seq mRNA LR workflow, cDNA was generated from SIRV-Set 4 (Lexogen) mRNA reference standards which contains both ERCC quantification controls and Long SIRV mRNA standards. 10 ng of mouse brain total RNA was prepared—with SIRV spike-ins added to account for approximately 5% of reads—using the workflow described in Figure 1. Libraries were sequenced by ONT MinION, FASTQ data was read-strand corrected using the Restrander tool, and data were aligned with minimap2. ERCC standard abundance for measured vs. theoretical concentration was plotted.
634377	SMART-Seq® mRNA Long Read	24 Rxns	USD $942.00
SMART-Seq mRNA Long Read generates full-length, barcoded cDNA directly from 1–1,000 intact cells or 10 pg–100 ng of total RNA in a convenient input volume of 1–9.5 µl. The full workflow can be completed within 2 days. 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 Library preparation workflow for the SMART-Seq mRNA Long Read kit. Library preparation workflow for the SMART-Seq mRNA Long Read kit. Starting with high-integrity RNA (RIN≥8) or intact cells, first-strand cDNA synthesis is primed by the SMART-Seq LR Oligo-dT and performed by an MMLV-derived reverse transcriptase (RT). Upon reaching the 5' end of each mRNA molecule, the RT adds nontemplated nucleotides to the first-strand cDNA. The SMART-Seq LR TSO contains a sequence that is complementary to the nontemplated nucleotides added by the RT. This primer hybridizes to the first-strand cDNA. In the template switching step, the RT uses the remainder of the SMART-Seq LR TSO as a template for the incorporation of an additional sequence on the end of the first-strand cDNA. The first-strand cDNA is then barcoded by forward and reverse primers from the SMART-Seq Long Read Index Kit (96 rxns) and amplified by the first PCR; a second PCR enriches for barcoded fragments. Samples are pooled and end-prepped, and sequencing adapters are ligated using the Ligation Sequencing Kit V14 (ONT). After sequencing, samples are basecalled and demultiplexed using ONT's Guppy. Downstream data analysis was performed using Cutadapt, Minimap2, SAMtools, Bedtools, and Salmon. Back SMART-Seq mRNA LR generates barcoded cDNA with a wide range of fragment lengths. SMART-Seq mRNA LR generates barcoded cDNA with a wide range of fragment lengths. SMART-Seq mRNA LR workflow was used to create cDNA from 10 pg or 10 ng total mouse brain RNA (n = 8). cDNA size distribution was measured on a 2100 Bioanalyzer (Agilent Technologies) using an Agilent High Sensitivity DNA Kit. Panel A. Barcoded cDNA was pooled per input, and libraries were generated using the Ligation Sequencing Kit V14. Libraries were sequenced on a MinION Flow Cell (ONT) for 72 hr. Samples were basecalled and demultiplexed using ONTs' Guppy and read-length distribution was plotted using MS-Excel. Panels B & C. Read-length distributions are shown for a representative sample of 10 pg (Panel B) and 10 ng (Panel C) total mouse brain RNA. Back SMART-Seq mRNA Long Read demonstrates high sensitivity across a broad range of inputs. SMART-Seq mRNA Long Read demonstrates high sensitivity across a broad range of inputs. To evaluate the performance, cDNA and sequencing libraries were generated from 10 pg and 10 ng total mouse brain RNA (MBR) using the SMART-Seq mRNA LR workflow. After sequencing, data was basecalled and demultiplexed using ONT's Guppy, and reads per barcode were downsampled to 400,000 reads. The read distribution for 10 pg and 10 ng MBR input. Panel A. is similar, showing consistent performance across the input range (n = 8 for 10 ng; n = 7 for 10 pg). Panel B. Reads of the 10 pg MBR dataset downsampled to assess gene and transcript sensitivity. Panel C. 10 ng MBR dataset downsampled to assess gene and transcript sensitivity. While neither of the two conditions (10 pg and 10 ng) were sequenced until saturation, both highlight the high gene and transcript sensitivity across read depths. Back SMART-Seq mRNA LR is highly reproducible across replicates for a broad range of inputs. SMART-Seq mRNA LR is highly reproducible across replicates for a broad range of inputs. The SMART-Seq mRNA LR workflow was used to create cDNA from 10 ng MBR of 96 replicates. Barcoded cDNA of all 96 samples were pooled, and libraries were generated and sequenced according to the workflow. Back SMART-Seq mRNA LR yields even gene-body coverage for ultra-low inputs. SMART-Seq mRNA LR yields even gene-body coverage for ultra-low inputs. 10 pg and 10 ng MBR samples underwent the SMART-Seq mRNA LR workflow. Gene-body coverage was assessed for the average of eight replicates of 10 pg and 10 ng MBR samples. Back SMART-Seq mRNA LR is compatible with direct cell inputs. SMART-Seq mRNA LR is compatible with direct cell inputs. cDNA was generated from single K562 cells or 1,000 K562 cells using the SMART-Seq mRNA LR workflow. After sequencing, data was basecalled and demultiplexed using Guppy, and reads per barcode were downsampled to 300,000 reads. Panel A. The average gene count for each condition is shown above the corresponding bar (n = 8 for single cell and n = 2 for 1,000 cells). Panel B. Pearson correlation was calculated from the gene matrix across eight single K562 cells. Panel C. Downsampling analysis of single K562 cells shows gene and transcript count per read depth. Back SMART-Seq mRNA LR detects full-length isoforms and gene fusions. SMART-Seq mRNA LR detects full-length isoforms and gene fusions. cDNA was generated using the SMART-Seq mRNA LR workflow. Basecalling and demultiplexing were performed using ONT's G uppy, and reads were aligned using Minimap2. Panels A & B. Isoforms of Snap25 (Panel A) and Nbr1 (Panel B) detected from 10 pg MBR input are visualized in IGV. Panel C. NUP214::XKR3 gene fusions detected from single-cell input are visualized in IGV. Back SMART-Seq mRNA LR is compatible with automation and miniaturization. SMART-Seq mRNA LR is compatible with automation and miniaturization. The SMART-Seq mRNA LR workflow was used to create cDNA from 10 ng MBR of 96 replicates manually on the benchtop with the full reaction volumes or automated at 1/8-volume on a mosquito HV (SPT Labtech). Barcoded cDNA of all 96 samples per experiment was pooled, and libraries were generated and sequenced for 72 hr according to the workflow. Samples were basecalled and demultiplexed using ONT Guppy and aligned with Minimap2. Panel A. Render of the mosquito HV liquid handler. Panel B. Gene counts were assessed with Salmon. Panel C. Mapping statistics of manually prepared cDNA compared to cDNA prepared using the mosquito HV. Back SMART-Seq mRNA LR performance with mRNA reference standards SMART-Seq mRNA LR performance with mRNA reference standards. To evaluate the performance of the SMART-Seq mRNA LR workflow, cDNA was generated from SIRV-Set 4 (Lexogen) mRNA reference standards which contains both ERCC quantification controls and Long SIRV mRNA standards. 10 ng of mouse brain total RNA was prepared—with SIRV spike-ins added to account for approximately 5% of reads—using the workflow described in Figure 1. Libraries were sequenced by ONT MinION, FASTQ data was read-strand corrected using the Restrander tool, and data were aligned with minimap2. ERCC standard abundance for measured vs. theoretical concentration was plotted. Back SMART-Seq mRNA LR performance with mRNA reference standards SMART-Seq mRNA LR performance with mRNA reference standards. To evaluate the performance of the SMART-Seq mRNA LR workflow, cDNA was generated from SIRV-Set 4 (Lexogen) mRNA reference standards which contains both ERCC quantification controls and Long SIRV mRNA standards. 10 ng of mouse brain total RNA was prepared—with SIRV spike-ins added to account for approximately 5% of reads—using the workflow described in Figure 1. Libraries were sequenced by ONT MinION, FASTQ data was read-strand corrected using the Restrander tool, and data were aligned with minimap2. IGV plots of data from 1 kb, 4 kb, 6 kb, and 8 kb long transcripts from the ERCC and long SIRV isoform set. Red colored reads indicate positive strand reads, blue indicates very rare negative-strand reads, and small purple or red marks indicate small indels/variations common in ONT-sequencing. Full-length is defined as reads that cover at least 90% of expected mRNA length. Back Visualization showing completeness of fragment lengths and strand orientation accuracy of the SSmRNA LR kit Visualization showing completeness of fragment lengths and strand orientation accuracy of the SSmRNA LR kit. This experiment used Spike-In RNA Variant (SIRV) controls (600–2,492 bp), showcasing detection of full-length fragments with a high percentage of complete transcripts, and accurate identification of strand orientation across all isoforms. Panel A. Coverage analysis of complete, full-length fragments. Panel B . Strand identification across different SIRV-spike-in species visualized via SIRVsuite. Back Automation- and miniaturization-friendly SSmRNA LR workflow comparison SMART-Seq mRNA LR performance with mRNA reference standards. To evaluate the performance of the SMART-Seq mRNA LR workflow, cDNA was generated from SIRV-Set 4 (Lexogen) mRNA reference standards which contains both ERCC quantification controls and Long SIRV mRNA standards. 10 ng of mouse brain total RNA was prepared—with SIRV spike-ins added to account for approximately 5% of reads—using the workflow described in Figure 1. Libraries were sequenced by ONT MinION, FASTQ data was read-strand corrected using the Restrander tool, and data were aligned with minimap2. ERCC standard abundance for measured vs. theoretical concentration was plotted.

Overview

Superior sensitivity and performance for ultra-low inputs—10 pg–100 ng total RNA or 1–1,000 intact cells
High-quality long-read sequencing data—reliably obtain average read lengths (N50) of ~2 kb, detecting full-length transcripts over 8 kb
Seamless integration of barcodes—multiplex up to 96 samples for single-tube long-read library preparation
Streamlined sample demultiplexing—convenient and efficient workflow for accurate sample assignment compatible with ONT's basecaller

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images/400-NGS/CC-TechFigs/SMART-Seq-mRNA-Nbr1-isoform.jpg

Additional product information

Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Product Components List to determine kit components. Certificates of Analysis and Product Components Lists are located under the Documents tab.

Enabling long-read RNA sequencing from low-input samples

Facilitating the discovery of complex isoforms and novel structural variants from 10 pg–100 ng of total RNA