TruSeq Stranded Total RNA with Ribo-Zero Globin

Prepare whole-transcriptome sequencing libraries from blood-derived RNA with an efficient workflow that removes ribosomal RNA and globin mRNA in a single step.

11.5 hr

Assay time

5.5 hr

Hands-on time

0.1 – 1 ug high-quality purified total RNA from blood

Input quantity

See full details in the specifications table

Overview

TruSeq Stranded Total RNA with Ribo-Zero Globin delivers a clear and comprehensive view of the transcriptome from blood-derived total RNA, with fast, efficient sequencing library preparation.

Key features

  • Uniform coverage and precise measurement of strand orientation

  • High-confidence discovery of alternative transcripts, antisense expression, and allele-specific expression

  • Discovery across both coding and multiple forms of noncoding RNA

High-quality whole-transcriptome analysis with precise strand information

These kits couple the benefits of Ribo-Zero ribosomal RNA reduction chemistry with RNA sequencing (RNA-Seq) technology for whole-transcriptome analysis of human, mouse, or rat samples. 

Efficient ribosomal RNA and globin mRNA removal in a single step

Whole-transcriptome analysis of blood-derived RNA requires the removal of two forms of abundant RNA—ribosomal RNA (both cytoplasmic and mitochondrial) as well as globin mRNA, which is present in high levels in whole blood.

Traditional removal methods require two independent steps. This means the need for additional reagents, a longer workflow, and more input RNA lost. TruSeq Stranded Total RNA with Ribo-Zero Globin leverages Ribo-Zero chemistry to efficiently remove both forms of abundant RNA in a single, rapid step.


Specifications


Required products

Illumina now offers modular product ordering to enable flexibility in your workflows. You may order any of the following library prep products with any of the following indexes.

/ Results

Applications

Example workflow


Project recommendations

Instrument Recommended number of samples Read length
NextSeq 550 System

RNA Profiling: 6-20 samples per run (based on 20 million reads per sample)
Transcriptome Analysis: 2-8 samples per run (based on 50 million reads per sample)

2 x 75 bp
2 x 75 bp

NovaSeq 6000 System

RNA Profiling (samples per run, dual flow cell): S1: 160, S2: 320, S4: 768. Limited by index combinations (dual). Based on 20 million reads.
Transcriptome Analysis (samples per run, dual flow cell): S1: 64, S2: 128, S4: 400. Limited by index combinations (dual). Based on 50 million reads. 

≤ 2 × 100 bp
≤ 2 × 100 bp


Related applications and methods

Compare

TruSeq Stranded Total RNA with Ribo-Zero Globin Illumina Stranded Total RNA Prep with Ribo-Zero Plus or Ribo-Zero Plus Microbiome
Assay time 11.5 hr ~7 hr
Automation capability Liquid Handling Robots Liquid Handling Robots
Content specifications Captures coding RNA plus multiple forms of non-coding RNA Captures coding RNA plus multiple forms of non-coding RNA
Description For whole-transcriptome sequencing studies of blood-derived RNA. Depletes cytoplasmic and mitochondrial rRNA plus globin mRNA.

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 5.5 hr < 3 hr
Input quantity 0.1 – 1 ug high-quality purified total RNA from blood

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 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 Bead-based rRNA depletion, cDNA synthesis, and PCR Enzymatic rRNA depletion, ligation-based addition of adapters and indexes
Method Whole-Transcriptome Sequencing Whole-Transcriptome Sequencing, Metatranscriptome Sequencing
Multiplexing High-throughput kits: Prepare 96 indexed samples using either combinatorial dual indexes or unique dual indexes, Low-throughput kits: Pool up to 12 samples. Or pool up to 24 samples with sets A and B together Up to 384 Unique Dual Indexes (UDIs)
Nucleic acid type RNA RNA
Specialized sample types Blood Microbiome Samples, Low-Input Samples, FFPE Tissue
Species category Mouse, Human, Rat Mouse, Human, Rat, Bacteria
Species details rRNA Removal: Depletes human, mouse, and rat rRNA (cytoplasmic and mitochondrial) and globin mRNA

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 

Technology Sequencing Sequencing
Variant class Single nucleotide polymorphisms (SNPs), Gene fusions, Novel transcripts, Transcript variants Single nucleotide polymorphisms (SNPs), Gene fusions, Novel transcripts, Transcript variants

Figures

Technical Replicates

Technical replicates of FFPE tissue show high concordance, indicating robust library prep performance. Axes are log2(FPKM). R2 value is shown.

Even Coverage Across Transcripts

TruSeq Stranded Total RNA gives excellent coverage across the top 1,000 expressed transcripts in both fresh-frozen (FF, top) and FFPE (bottom) tumor and matched normal breast tissue, with > 98% aligned stranded reads. X-axis: position along transcript, Y-axis = percent coverage of combined reads.

Library prep (2)

TruSeq® Stranded Total RNA Library Prep Globin (48 Samples)

20020612

Includes RiboZero beads to deplete rRNA and globin mRNA from blood samples, PCR master mix to transcript RNA into cDNA, and reagents for preparing 48 samples. Purchase index adapters and Purification Beads separately.

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TruSeq® Stranded Total RNA Library Prep Globin (96 Samples)

20020613

Includes RiboZero beads to deplete rRNA and globin mRNA from blood samples, PCR master mix to transcript RNA into cDNA, and reagents for preparing 48 samples. Purchase index adapters and Purification Beads separately.

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Index adapters (4)

TruSeq RNA CD Index Plate (96 Indexes, 96 Samples)

20019792

TruSeq Stranded RNA HT Kit 96 Samples Adapter Plate Box

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IDT for Illumina – TruSeq RNA UD Indexes (24 Indexes, 96 Samples)

20020591

New Unique Dual Indexes (UDIs), compatible with TruSeq Stranded mRNA and TruSeq Stranded Total RNA Library Prep Kits. The UDI kit is provided in a plate format, contains 24 UDIs (x4), sufficient for 96 samples.

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IDT for Illumina – TruSeq DNA UD Indexes v2 (96 Indexes, 96 Samples)

20040870

Includes 96, 8 bp indexes sufficient for labeling 96 samples and is compatible with TruSeq Nano and TruSeq PCR-Free Library Prep Kits. Version 2 contains improvements for 8 of the 192 index oligos in plate 20022370, with no changes to the remaining 184. Purchase library preps and probe panels separately.

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IDT for Illumina – TruSeq RNA UD Indexes v2 (96 Indexes, 96 Samples)

20040871

Includes 96, 8 bp indexes sufficient for labeling 96 samples and is compatible with TruSeq Stranded mRNA and TruSeq Stranded Total RNA Library Prep Kits. Version 2 contains improvements for 8 of the 192 index oligos in plate 20022371, with no changes to the remaining 184. Purchase library preps and probe panels separately.

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Selection summary

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Illumina DNA Prep

A fast, integrated workflow for preparing libraries for use in a wide range of sequencing applications.

Limited Use Label License:

The TruSeq Stranded Total RNA with Ribo-Zero Globin Workflow product and its use are the subject of one or more issued and/or pending U.S. and foreign patent applications owned by Max Planck Gesellschaft, exclusively licensed to New England Biolabs, Inc. and sublicensed to Illumina, Inc. The purchase of this product from Illumina, Inc., its affiliates, or its authorized resellers and distributors conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product by the buyer (whether the buyer is an academic or for profit entity). The purchase of this product does not convey a license under any claims in the foregoing patents or patent applications direct to producing the product. The buyer cannot sell or otherwise transfer this product or its components to a third party or otherwise use the product for the following COMMERICAL PURPOSES: (I) use of the product or its components in manufacturing; or (2) use of the product or its components for therapeutic or prophylactic purposes in human or animals.

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