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Illumina Stranded Total RNA Prep with Ribo-Zero Plus or Ribo-Zero Plus Microbiome

Streamlined RNA-Seq solution for clear and comprehensive analysis across the coding and non-coding transcriptome with exceptional study flexibility.

~7 hr

Assay time

< 3 hr

Hands-on time

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

Input quantity

See full details in the specifications table

Overview

Illumina Total RNA Prep with Ribo-Zero Plus provides accurate measurement of gene and transcript abundance and detects both known and novel features in coding and multiple forms of noncoding RNA. Researchers can have confidence in discovery of alternative transcripts, gene fusions, and allele-specific expression. Additionally, the Illumina Stranded Total RNA Prep with Ribo-Zero Plus Microbiome delivers high-quality data for metatranscriptomics applications.

Key features

  • Exceptional performance—Removes prokaryotic and eukaryotic ribosomal RNA (rRNA) and globin RNA from multiple species in a single reaction to allow rich transcriptome analysis

  • High-quality data—Achieves high-sensitivity from low RNA inputs (1–1000 ng) and difficult sample types, including formalin-fixed paraffin-embedded (FFPE), stool samples, and other low-quality samples

  • Fast, flexible workflow—Prepares libraries 40% faster than comparable kits. Innovations to the workflow, including shorter incubation times and reduced sample cleanup steps, result in a total assay time of 7 hr

Specifications

Assay time ~7 hr
Automation capability Liquid handling robots
Content specifications Captures coding RNA plus multiple forms of non-coding RNA
Description 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
Input quantity 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
Mechanism of action Enzymatic rRNA depletion, ligation-based addition of adapters and indexes
Method Whole-transcriptome sequencing, Metatranscriptome sequencing
Multiplexing Up to 384 Unique Dual Indexes (UDIs)
Nucleic acid type RNA
Specialized sample types Microbiome samples, Low-input samples, FFPE tissue
Species category Mouse, Human, Rat, Bacteria
Species details 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
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.

Indexes are sold separately and required for multiplexing. Choose the indexing option that best fits your target sequencing throughput. Sequencing throughput and multiplexing capabilities can vary depending on sequencer and flow cell reagents.

Learn more about multiplexing

/ Results

Applications

Illumina Stranded Total RNA Prep with Ribo-Zero Plus or Ribo-Zero Plus Microbiome kits enable preparation of whole-transcriptome sequencing-ready libraries from low RNA inputs and low-quality samples in approximately 7 hr, with less than 3 hr hands-on time.

Example workflow

1
Prep

Illumina Stranded Total RNA Prep with Ribo-Zero Plus or Ribo-Zero Plus Microbiome

2
Sequence
3
Analyze

Project recommendations

Instrument Recommended number of samples Read length
NextSeq 550 System

Mid output: 2 samples per flow cell 

High output: 8 samples per flow cell 

2 × 75 bp
NextSeq 2000 System

P2: 8 samples per flow cell 

P3: 24 samples per flow cell

2 × 75 bp
NovaSeq 6000 System

SP: 16 samples per flow cell 

S1: 32 samples per flow cell

S2: 82 samples per flow cell 

S4: 200 samples per flow cell 

2 × 75 bp

Related applications and methods

Total RNA sequencing

Analyze both coding RNA and multiple forms of noncoding RNA for a comprehensive view of the transcriptome.

Microbial transcriptomics

RNA sequencing (RNA-Seq) offers an unbiased approach for analyzing and quantifying bacterial, viral, and other microbial transcripts.

Gene expression analysis

Learn how to profile gene expression changes for a deeper understanding of biology.

How others use this product

Capturing dynamic shifts in the microbiome with metatranscriptomics

Documentation

Product literature
Support documentation

Compare

Illumina Stranded Total RNA Prep with Ribo-Zero Plus or Ribo-Zero Plus Microbiome TruSeq Stranded Total RNA TruSeq Stranded Total RNA with Ribo-Zero Globin
Assay time ~7 hr 11.5 hr 11.5 hr
Automation capability Liquid handling robots 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 Captures coding RNA plus multiple forms of non-coding RNA
Description 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.  For whole-transcriptome sequencing studies. The Total RNA version depletes cytoplasmic rRNA while the Total RNA Gold version depletes cytoplasmic and mitochondrial rRNA. For whole-transcriptome sequencing studies of blood-derived RNA. Depletes cytoplasmic and mitochondrial rRNA plus globin mRNA.
Hands-on time < 3 hr 5.5 hr 5.5 hr
Input quantity 1–1000 ng total RNA for standard-quality RNA samples. 10 ng total RNA minimum recommended for optimal performance and FFPE samples  0.1 – 1 ug high-quality total RNA. Lower-quality samples may require further optimization. 0.1 – 1 ug high-quality purified total RNA from blood
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 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 Enzymatic rRNA depletion, ligation-based addition of adapters and indexes Bead-based rRNA depletion, cDNA synthesis, and PCR Bead-based rRNA depletion, cDNA synthesis, and PCR
Method Whole-transcriptome sequencing, Metatranscriptome sequencing Whole-transcriptome sequencing Whole-transcriptome sequencing
Multiplexing Up to 384 Unique Dual Indexes (UDIs) Low-throughput kits: Pool up to 12 samples. Or pool up to 24 samples with sets A and B together, High-throughput kits: Prepare 96 indexed samples using either combinatorial dual indexes or unique dual indexes 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
Nucleic acid type RNA RNA RNA
Specialized sample types Microbiome samples, Low-input samples, FFPE tissue FFPE tissue Blood
Species category Mouse, Human, Rat, Bacteria Mouse, Human, Rat Mouse, Human, Rat
Species details 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  rRNA Removal: Depletes human, mouse, and rat rRNA rRNA Removal: Depletes human, mouse, and rat rRNA (cytoplasmic and mitochondrial) and globin mRNA
Strand specificity Stranded
Technology 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

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Figures

Illumina Stranded Total RNA Prep with Ribo-Zero Plus workflow

Illumina Stranded Total RNA 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.

Comparison of performance metrics

Illumina Stranded Total RNA Prep with Ribo-Zero Plus was compared against TruSeq Stranded Total RNA with Ribo-Zero. Illumina Stranded Total RNA Prep with Ribo-Zero Plus was more effective at rRNA depletion, particularly at low inputs of 10 ng and 1 ng total UHR RNA. 

Robust ribodepletion with the Ribo-Zero Plus Microbiome rRNA Depletion kit

The Ribo-Zero Plus Microbiome rRNA Depletion kit effectively depletes rRNA (5S, 16S, and 23S) levels in stool samples. Results are compared to Ribo-Zero Plus rRNA Depletion Kit and a commercially available ribodepletion kit from Company X using two stool sample mixtures (Sample 1 and Sample 2).

RNA species targeted for reduction

The Illumina Stranded Total RNA Prep kit includes (A) Ribo-Zero Plus for removing abundant rRNA from multiple species, including human, mouse, rat, bacteria, and epidemiology samples, or the (B) Ribo-Zero Plus Microbiome which provides robust depletion of abundant rRNA in complex microbial samples, including stool samples, for metatranscriptomic studies.

A. Ribo-Zero Plus kit

Depletion Target rRNA Target
Human cytoplasmic rRNA 28S, 18S, 5.8S, and 5S
Human mitochondrial rRNA 12S, and 16S
Human beta globin transcripts HBA1, HBA2, HBB, HBG1, and HBG2
Mouse and rat 12S, 16S, 28S, 18S, 5.8S, and 5S
Gram(-) bacterial rRNAs E.coli: 5S, 16S, 23S
>Gram(+) bacterial rRNAs B. subtilis: 5S, 16S, 23S

B. Ribo-Zero Plus Microbiome kit

Depletion Target rRNA Target
Microbiome rRNA* 5S, 16S, 23S from diverse microbes including common adult, infant bacterial species and ATCC MSA-2002, MSA-2005, and MSA-2006

*Only included in Illumina Stranded Total Ribo-Zero Plus Microbiome kit (Cat#20072063). The rRNA depletion was optimized for human microbiome rRNA, performance can vary when using other sample types.

Resources

Unbiased microbial profiling with metatranscriptomics

See how Drs. Tonya Ward, Emily Hollister, and Le François of Diversigen and DNA Genotek developed workflows for metatranscriptomic studies and read their views for this research area’s potentially impactful future.

Read customer spotlight

Metatranscriptomics: challenges and opportunities

In this webinar, you'll hear from three leading experts in the field as they share their insights and key considerations on sample collection and processing, rRNA depletion, and bioinformatic analysis.

Watch webinar

Library prep (3)

Illumina® Stranded Total RNA Prep, Ligation with Ribo-Zero Plus (16 Samples)

20040525

Includes reagents and Ribo-Zero Plus depletion module for preparing 16 samples. Compatible only with IDT for Illumina RNA UD indexes, Ligation. Indexes and Purification Beads sold separately.

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Illumina® Stranded Total RNA Prep, Ligation with Ribo-Zero Plus (96 Samples)

20040529

Includes reagents and Ribo-Zero Plus depletion module for preparing 96 samples. Compatible only with IDT for Illumina RNA UD indexes, Ligation. Indexes and Purification Beads sold separately.

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Illumina® Stranded Total RNA Prep with Ligation, Ribo-Zero Plus Microbiome (96 Samples)

20072063

Includes reagents and Ribo-Zero Plus Microbiome depletion module for preparing 96 libraries. Compatible only with IDT for Illumina RNA UD indexes, Ligation. Indexes sold 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 Total RNA Library Preparation Kit Training - Customer Site

20044764

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

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FAQs

Illumina Stranded Total RNA Prep with Ligation, Ribo-Zero Plus Microbiome kit contains expanded capture probes that target 5S, 16S, and 23S rRNA from diverse microbial species, including common adult and infant gut bacteria, while the Ribo-Zero Plus rRNA Depletion Kit targets the 5S, 16S, and 23S of E. coli and B. subtilis.

Illumina Stranded Total RNA Prep with Ligation, Ribo-Zero Plus Microbiome depletes human globin transcripts and rRNAs from multiple species, including human, mouse, rat, and diverse strains of bacteria commonly found in human gut and skin. Illumina cannot guarantee rRNA removal of species outside of those listed.

The Universal Human Reference RNA positive sample control (Catalog no. 740000) from Agilent Technologies is recommended for use with Illumina Stranded Total RNA Prep.

To check the quality and concentration of the final libraries, the following two methods can be used:

  • If using the NextSeq 1000/2000 Sequencing System, dilute 1 μl library 1:10,000 dilution, and then analyze the library using KAPA qPCR library quantification kit

  • For all other sequencing systems, analyze 1 µl library using the Agilent 2100 Bioanalyzer and DNA 1000 Kit

The Illumina Stranded Total RNA Prep targets the strand of origin.

The minimum recommended input amount of RNA is 25 ng for optimal performance when using the Illumina Stranded Total RNA Prep with Ligation, Ribo-Zero Plus Microbiome kit.

/ Results

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