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Advanced chemistry, optics, and informatics combine to deliver exceptional speed and data quality, outstanding throughput and scalability.
Streamlined RNA-Seq solution for clear and comprehensive analysis across the coding and non-coding transcriptome with exceptional study flexibility.
Assay time
Hands-on time
Input quantity
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.
Assay time | ~7 hr |
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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 |
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.
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.
Illumina Stranded Total RNA Prep with Ribo-Zero Plus or Ribo-Zero Plus Microbiome
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 |
Analyze both coding RNA and multiple forms of noncoding RNA for a comprehensive view of the transcriptome.
Learn how to profile gene expression changes for a deeper understanding of biology.
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 | |||
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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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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.
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.
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).
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.
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.
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.
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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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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.
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:
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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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.
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