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

See full details in the specifications table

Overview

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.

Key features

  • Precise measurement of strand orientation—Enables detection of antisense transcription, enhances transcript annotation, and increases alignment efficiency

  • Exceptional performance—Provides excellent polyA capture efficiency and coverage uniformity, minimizing required sequencing depth for accurate, unbiased detection of the coding transcriptome.

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  • High-quality data from low-input samples—Achieves highly sensitive results from as little as 25 ng of high-quality RNA*

  • Fast library preparation workflow—Prepares libraries ~40% faster than TruSeq Stranded mRNA; shorter incubation times and reduced sample cleanup steps result in a total assay time of 6.5 hr

*Not compatible with FFPE samples


Specifications


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.

/ Results

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


Project recommendations

Instrument Recommended number of samples Read length
NextSeq 550 System

Mid output: 5
High output: 16
(based on 25M reads per sample)

2 × 75 bp

NextSeq 2000 System

P2: 16
P3: 48
(based on 25M reads per sample)

2 × 75 bp

NovaSeq 6000 System

SP: 32
S1: 64
S2: 164
S4: 384
(based on 25M reads per sample)

2 × 75 bp



Related applications and methods

Documentation

Product literature

Compare

Illumina Stranded mRNA Prep TruSeq Stranded mRNA Illumina RNA Prep with Enrichment Illumina Stranded Total RNA Prep with Ribo-Zero Plus or Ribo-Zero Plus Microbiome
Assay time 6.5 hr ~10.5 hr < 9 hr ~7 hr
Automation capability Liquid handling robots Liquid handling robots Liquid handling robots Liquid handling robots
Automation details Explore available automation methods Explore available automation methods
Content specifications Captures the coding transcriptome with strand information Captures the coding transcriptome with strand information Captures the coding transcriptome when used with Illumina Exome Panel Captures coding RNA plus multiple forms of non-coding RNA
Description A simple, cost-effective solution for analysis of the coding transcriptome with precise strand information Gives researchers a clear, comprehensive view of the coding transcriptome with precise strand information. A reproducible, economical solution enabling targeted transcript detection and discovery from a broad range of sample types and inputs including formalin-fixed, paraffin- embedded (FFPE) tissues and other low-quality samples

For the preparation of whole-transcriptome sequencing-ready libraries from low RNA inputs and low-quality samples that supports a wide range of RNA-Seq applications. Includes Ribo-Zero Plus for single-tube depletion of rRNA and globin RNA from multiple species to facilitate rich transcriptome analyses. 

Hands-on time < 3 hr ~4.5 hr < 2 hr < 3 hr
Input quantity 25-1000 ng standard-quality total RNA 0.1 – 1 ug total RNA or 10 - 100 ng previously isolated mRNA (from species with polyA tails) 10 ng RNA; 20 ng FFPE RNA

1–1000 ng total RNA for standard-quality RNA samples. 10 ng total RNA minimum recommended for optimal performance and FFPE samples 

Instruments NextSeq 550 System, NextSeq 2000 System, NextSeq 1000 System, NovaSeq X System, NextSeq 500 System, NovaSeq 6000 System, NovaSeq X Plus System MiSeq System, NextSeq 550 System, NextSeq 2000 System, NextSeq 1000 System, NovaSeq X System, NextSeq 500 System, NovaSeq 6000 System, NovaSeq X Plus System MiSeq System, iSeq 100 System, NextSeq 550 System, NextSeq 2000 System, NextSeq 1000 System, MiniSeq System, NovaSeq 6000 System NextSeq 550 System, NextSeq 2000 System, NextSeq 1000 System, NovaSeq X System, NextSeq 500 System, NovaSeq 6000 System, NovaSeq X Plus System
Mechanism of action PolyA capture, ligation-based addition of adapters and indexes Oligo-dT beads capture polyA tails Bead-linked transposome Enzymatic rRNA depletion, ligation-based addition of adapters and indexes
Method mRNA sequencing mRNA sequencing mRNA sequencing, Targeted RNA sequencing, Target enrichment Whole-transcriptome sequencing, Metatranscriptome sequencing
Multiplexing Up to 384 Unique Dual Indexes (UDIs) 1–96 Up to 384 Unique Dual Indexes (UDIs) Up to 384 Unique Dual Indexes (UDIs)
Nucleic acid type RNA RNA RNA RNA
Specialized sample types Not FFPE-Compatible, Low-Input Samples Not FFPE-Compatible Blood, Low-Input Samples, FFPE Tissue Microbiome Samples, Low-Input Samples, FFPE Tissue
Species category Mammalian, Bovine, Mouse, Human, Rat Mammalian, Bovine, Mouse, Human, Rat Human, Virus Mouse, Human, Rat, Bacteria
Species details Works with high-quality RNA from any species with polyA tails Works with high-quality RNA from any species with polyA tails

Ribo-Zero Plus depletes abundant transcripts from multiple species including: human cytoplasmic and mitochondrial rRNA, mouse rRNA, rat rRNA, bacteria (E. coli and B. subtilis) rRNA, and human beta globin transcripts 

Ribo-Zero Plus Microbiome depletes abundant transcripts from multiple species including: those depleted in Ribo-Zero Plus as well as diverse microbial species and ATCC MSA-2002, MSA-2005, and MSA-2006 

Strand specificity Stranded Stranded Non-stranded 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.
Technology Sequencing Sequencing Sequencing Sequencing
Variant class Single nucleotide polymorphisms (SNPs), Gene fusions, Novel transcripts, Transcript variants Single nucleotide polymorphisms (SNPs), Gene fusions, Novel transcripts, Transcript variants Single nucleotide polymorphisms (SNPs), Gene fusions, Novel transcripts, Transcript variants Single nucleotide polymorphisms (SNPs), Gene fusions, Novel transcripts, Transcript variants

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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.

/ Results

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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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.

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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.

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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.

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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.

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Services (1)

Illumina Stranded mRNA LP Kit Training - Customer Site

20044765

2-day on-site training for the Illumina Stranded mRNA Library Preparation Kit. Hands-on training includes quantification of mRNA input as well as best practices, troubleshooting tips, and training on Illumina-supported analysis tools specific to the application workflow.

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Selection summary

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Qty

Unit Price

/ Results

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