Illumina Stranded mRNA Prep provides a streamlined RNA sequencing (RNA-Seq) solution for clear and comprehensive analysis across the coding transcriptome for discovery of features such as novel isoforms, gene fusions, and allele-specific expression.
Illumina Stranded mRNA Prep
A rapid, cost-effective workflow for accurate, unbiased detection of the protein-coding transcriptome with precise measurement of strand information.
6.5 hr
Assay time
< 3 hr
Hands-on time
25-1000 ng standard-quality total RNA
Input quantity
Overview
Key features
*Not compatible with FFPE samples
Specifications
| Assay time | 6.5 hr |
|---|---|
| Automation capability | Liquid Handling Robots |
| Automation details | Explore available automation methods |
| Content specifications | Captures the coding transcriptome with strand information |
| Description | A simple, cost-effective solution for analysis of the coding transcriptome with precise strand information |
| Hands-on time | < 3 hr |
| Input quantity | 25-1000 ng standard-quality total RNA |
| Mechanism of action | PolyA capture, ligation-based addition of adapters and indexes |
| Method | mRNA Sequencing |
| Multiplexing | Up to 384 Unique Dual Indexes (UDIs) |
| Nucleic acid type | RNA |
| Specialized sample types | Not FFPE-Compatible, Low-Input Samples |
| Species category | Mammalian, Bovine, Mouse, Human, Rat |
| Species details | Works with high-quality RNA from any species with polyA tails |
| Strand specificity | Stranded |
| System compatibility details | Library prep is designed to be compatible with all Illumina sequencing systems, and is extensively validated on the NextSeq 500/550 and NovaSeq 6000 Systems. |
| Systems | NextSeq 2000 System, NextSeq 1000 System, NovaSeq X System, NextSeq 500 System, NovaSeq 6000 System, NovaSeq X Plus System, NextSeq 550 System |
| Technology | Sequencing |
| Variant class | Single Nucleotide Polymorphisms (SNPs), Gene Fusions, Novel Transcripts, Transcript Variants |
Required products
Choose the library prep kit size (16 sample or 96 sample) that meets your processing needs.
Unique dual indexes are sold separately and required for multiplexing. Choose the indexing option that best fits your target sequencing throughput. You can add Set A, B, C, and D together to sequence up to 384 samples in a single run. Sequencing throughput and multiplexing capabilities can vary depending on sequencer and flow cell reagents.
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Applications
Illumina Stranded mRNA Prep enables preparation of sequencing-ready transcriptome libraries in a single day with low sample inputs of standard (non-degraded) RNA from a wide range of sample types.
Example workflow
Prep
Illumina Stranded mRNA Prep
Project recommendations
| Instrument | Recommended number of samples | Read length |
|---|---|---|
| NextSeq 550 System | Mid output: 5 |
2 × 75 bp |
| NextSeq 2000 System | P2: 16 |
2 × 75 bp |
| NovaSeq 6000 System | SP: 32 |
2 × 75 bp |
Featured application notes
Related applications and methods
mRNA sequencing (mRNA-Seq) using NGS can identify both known and novel transcripts, and measure transcript abundance.
Learn how to analyze transcriptome changes or profile genome-wide gene expression levels in a single experiment with next-generation sequencing methods.
Drug response RNA biomarker analysis
Assessment of RNA-based biomarkers predictive of therapeutic response is increasingly becoming an integral part of the drug development process.
Documentation
Product literature
Support documentation
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Figures
Comparison of performance metrics
(Left) Performance metrics for Illumina Stranded mRNA Prep. (Right) Illumina Stranded mRNA Prep was compared against TruSeq Stranded mRNA. Illumina Stranded mRNA Prep showed superior performance, particularly with an input of 25 ng UHR RNA.
Greater gene discovery at low input
Illumina Stranded mRNA Prep enables greater gene detection with low RNA inputs, as compared to TruSeq Stranded mRNA. The number of genes detected is reported at 30M subsampled paired-end reads PF. More genes detected at 1× coverage is an indicator of greater sensitivity.
High concordance with legacy kit
Illumina Stranded mRNA Prep produces highly concordant data with TruSeq Stranded mRNA at (A) equivalent inputs of 100 ng UHR RNA and (B) with reduced input of 25 ng vs 100 ng UHR RNA.
High data concordance
Illumina Stranded mRNA Prep achieves high data concordance between (A) technical replicates of 25 ng UHR RNA and (B) between input amounts of 25 ng and 100 ng UHR RNA. Libraries were sequenced at 2 × 74 bp, subsampled to 30M reads. Data analysis was performed using the BaseSpace RNA-Seq Alignment App.
Illumina Stranded mRNA Prep workflow
Illumina Stranded mRNA Prep delivers a fast workflow with reduced hands-on time. Times may vary depending on equipment used, number of samples processed, automation procedures, or user experience.
FAQs
To accurately determine gene expression from overlapping genes, it is important to retain the information found on the strand of origin. Stranded RNA-Seq allows the first and second cDNA strands to be distinguished so that the second strand can be degraded while the first cDNA strand (strand of origin) will undergo further PCR amplification.
Library preparation takes 6.5 hr with less than 3 hr of hands-on time.
Oligo(dT) magnetic beads capture and purify the mRNA containing polyA tails. The purified mRNA is fragmented and copied into first-strand cDNA using reverse transcriptase and random primers. In a second-strand cDNA synthesis step, dUTP replaces dTTP to achieve strand specificity. The final steps add adenine and thymine bases to fragment ends and ligate adapters. The resulting products are purified and selectively amplified for sequencing.
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Library prep (2)
Illumina® Stranded mRNA Prep, Ligation (16 Samples)
20040532
Includes reagents for preparing and enriching 16 libraries (16, 1-plex enrichment reactions). Purchase enrichment probe panel, Purification Beads and index adapters separately.
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Illumina® Stranded mRNA Prep, Ligation (96 Samples)
20040534
Includes reagents for preparing and enriching 96 libraries (32, 3-plex enrichment reactions). Purchase enrichment probe panel, Purification Beads and index adapters separately.
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Index adapters (8)
Illumina® RNA UD Indexes Set A, Ligation (96 Indexes, 96 Samples)
20091655
Illumina® RNA UD Indexes Set A, Ligation (96 Indexes, 96 Samples) Includes 96, 10 bp indexes sufficient for labeling 96 samples and RNA Index Anchor plate. Used with Illumina Stranded Total RNA Prep and Illumina Stranded mRNA Prep Kits. Purchase library prep reagents separately.
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Illumina® RNA UD Indexes Set B, Ligation (96 Indexes, 96 Samples)
20091657
Illumina® RNA UD Indexes Set B, Ligation (96 Indexes, 96 Samples) Includes 96, 10 bp indexes sufficient for labeling 96 samples and RNA Index Anchor plate. Used with Illumina Stranded Total RNA Prep and Illumina Stranded mRNA Prep Kits. Purchase library prep reagents separately.
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Illumina® RNA UD Indexes Set C, Ligation (96 Indexes, 96 Samples)
20091659
Illumina® RNA UD Indexes Set C, Ligation (96 Indexes, 96 Samples) Includes 96, 10 bp indexes sufficient for labeling 96 samples and RNA Index Anchor plate. Used with Illumina Stranded Total RNA Prep and Illumina Stranded mRNA Prep Kits. Purchase library prep reagents separately.
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Discounts:
Illumina® RNA UD Indexes Set D, Ligation (96 Indexes, 96 Samples)
20091661
Illumina® RNA UD Indexes Set D, Ligation (96 Indexes, 96 Samples) Includes 96, 10 bp indexes sufficient for labeling 96 samples and RNA Index Anchor plate. Used with Illumina Stranded Total RNA Prep and Illumina Stranded mRNA Prep Kits. Purchase library prep reagents separately.
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IDT® for Illumina® RNA UD Indexes Set A, Ligation (96 Indexes, 96 Samples)
20040553
Includes 96, 10 bp indexes sufficient for labeling 96 samples and RNA Index Anchor plate. Used with Illumina Stranded Total RNA Prep and Illumina Stranded mRNA Prep Kits. Purchase library prep and probe panels separately.
List Price:
Discounts:
IDT® for Illumina® RNA UD Indexes Set B, Ligation (96 Indexes, 96 Samples)
20040554
Includes 96, 10 bp indexes sufficient for labeling 96 samples and RNA Index Anchor plate. Used with Illumina Stranded Total RNA Prep and Illumina Stranded mRNA Prep Kits. Purchase library prep and probe panels separately.
List Price:
Discounts:
IDT® for Illumina® RNA UD Indexes Set C, Ligation (96 Indexes, 96 Samples)
20040555
Includes 96, 10 bp indexes sufficient for labeling 96 samples and RNA Index Anchor plate. Used with Illumina Stranded Total RNA Prep and Illumina Stranded mRNA Prep Kits. Purchase library prep and probe panels separately.
List Price:
Discounts:
IDT® for Illumina® RNA UD Indexes Set D, Ligation (96 Indexes, 96 Samples)
20040556
Includes 96, 10 bp indexes sufficient for labeling 96 samples and RNA Index Anchor plate. Used with Illumina Stranded Total RNA Prep and Illumina Stranded mRNA Prep Kits. Purchase library prep and probe panels separately.
List Price:
Discounts:
Services (1)
Illumina Stranded mRNA Library Prep Training
20044765
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