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
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
Illumina Stranded Total RNA Prep with Ribo-Zero Plus or Ribo-Zero Plus Microbiome
NextSeq 550 System
NextSeq 1000 and 2000 Systems
NovaSeq 6000 System
NovaSeq X Series
DRAGEN RNA Pipeline
RNA-Seq Differential Expression
BaseSpace Microbiome Metatranscriptomics app
Project recommendations
Instrument: Recommended number of samples
NextSeq 550 System: Mid output: 2 samples per flow cell High output: 8 samples per flow cell
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
P2: 8 samples per flow cell
P3: 24 samples per flow cell
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
SP: 16 samples per flow cell
S1: 32 samples per flow cell
S2: 82 samples per flow cell
S4: 200 samples per flow cell
Related applications and methods
Total RNA sequencing
Analyze both coding RNA and multiple forms of noncoding RNA for a comprehensive view of the transcriptome.
Metatranscriptome sequencing
Explore the vast potential of microbial community gene sequencing for health and disease research.
Gene expression analysis
Learn how to profile gene expression changes for a deeper understanding of biology.
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
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.
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.
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.
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. View less
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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. View less
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. View less
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. View less
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. View less
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. View less
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. View less
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. View less
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How is the Ribo-Zero Plus Microbiome kit different from the Ribo-Zero kit?
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 .
Does Illumina Stranded Total RNA Prep with Ribo-Zero Plus Microbiome deplete globin mRNA from non-human total RNA?
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.
Which positive control is recommended for use with Illumina Stranded Total RNA Prep?
The Universal Human Reference RNA positive sample control (Catalog no. 740000) from Agilent Technologies is recommended for use with Illumina Stranded Total RNA Prep.
What is the recommended QC method for Illumina Stranded Total RNA Prep libraries?
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
Which RNA molecules does Illumina Stranded Total RNA Prep target?
The Illumina Stranded Total RNA Prep targets the strand of origin.
What is the minimum input for Illumina Stranded Total RNA Prep with Ribo-Zero Plus Microbiome?
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.
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