Illumina Stranded Total RNA Library Prep Training
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Illumina Total RNA Prep with Ribo-Zero Plus provides exceptional performance for the analysis of coding and multiple forms of noncoding RNA. Gain high-confidence discovery of alternative transcripts, gene fusions, and allele-specific expression.
Illumina Total RNA Prep with Ribo-Zero Plus is based on TruSeq ligation technology, which has been cited in over 9,926 publications since 2011. This ligation method results in high coverage uniformity, precise strand information, and robust and reliable library preparation even from degraded samples.
Learn more about the technology
Illumina公司总RNA与Ribo-Zero + suppor预科ts a broad range of RNA inputs, from 1 ng to 1000 ng*. It’s compatible with variable sample types, including formalin-fixed paraffin-embedded (FFPE) and other low-quality samples. The included Ribo-Zero Plus removes abundant RNA from multiple species, including human, mouse, rat, bacteria, and epidemiology samples.
Prepare libraries in as little as 7 hours. Illumina-qualified automation methods will be available soon through ourautomation partners.
Multiplex up to 384 samples and sequence in a single run. (Index Sets A and B are available now; Sets C and D are coming soon.) The number of samples sequenced per run depends on the throughput capacity of the sequencer.
*This input range is applicable to high-quality RNA. We recommend 10 ng input for best results and for FFPE or degraded samples.
| Instrument | Recommended Number of Samples | Read Length |
|---|---|---|
| NextSeq 550 System | Mid output: 2 High output: 8 (基于50 m读取/样本) |
2 × 75 bp |
| NextSeq 2000 System | Whole Transcriptome Analysis: 8 (P2)-20 (P3) samples per run (based on 50M reads per sample) |
2 × 75 bp |
| NovaSeq 6000 System | Whole Transcriptome Analysis: SP: 16 S1: 32 S2: 82 S4: 200 samples per run (based on 50M reads per sample) |
2 × 75 bp |
| Illumina Stranded Total RNA Prep with Ribo-Zero Plus | RN TruSeq困总A | RN TruSeq困总A with Ribo-Zero Globin | |
|---|---|---|---|
| Assay Time | ~7 hours | 11.5 hours | 11.5 hours |
| 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 | 对于whole-transcriptome sequencing studies of multiple sample types. Includes Ribo-Zero plus module for single-tube depletion of abundant transcripts from multiple species | 对于whole-transcriptome sequencing studies. The Total RNA version depletes cytoplasmic rRNA while the Total RNA Gold version depletes cytoplasmic and mitochondrial rRNA. | 对于whole-transcriptome sequencing studies of blood-derived RNA. Depletes cytoplasmic and mitochondrial rRNA plus globin mRNA. |
| Hands-On Time | < 3 hours | 5.5 hours | 5.5 hours |
| Input Quantity | 1-1000 ng total RNA for standard-quality RNA samples. 10ng 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 |
| 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 | 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 indexes samples using either combinatorial dual indexes or unique dual indexes (available soon) | High-throughput kits: Prepare 96 indexes samples using either combinatorial dual indexes or unique dual indexes (available soon), Low-throughput kits: Pool up to 12 samples. Or pool up to 24 samples with sets A and B together |
| Species Category | Bacteria, Human, Mouse, Rat, Virus | Human, Mouse, Rat | Human, Mouse, Rat |
| Technology | Sequencing | Sequencing | Sequencing |
Top- Multispecies ribodepletion with Illumina Stranded Total RNA Prep with Ribo-Zero Plus. Illumina Stranded Total RNA Prep with Ribo-Zero Plus effectively depletes rRNA levels for human, mouse, rat, and bacterial species in a single-tube reaction. Results are compared to TruSeq Stranded Total RNA paired with Ribo-Zero Gold for mammalian species and Ribo-Zero Bacteria for E. coli (B. subtilis data not shown).Bottom- Globin mRNA depletion from human peripheral blood leukocytes with Illumina Stranded Total RNA Prep with Ribo-Zero Plus
Illumina Stranded Total RNA Prep provides high coverage uniformity for (A) high-quality and synthetically degraded UHR RNA (RIN=2) and (B) FFPE RNA at input levels of 100 ng and 10 ng. The FFPE sample had a DV200 quality score of 55%. All libraries were sequenced on a NovaSeq 6000 System at 50M reads. Data analysis was performed using the BaseSpace RNA-Seq Alignment App v2.0.1.
Top- 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, particularly at low inputs of 10 ng and 1 ng total UHR RNA. Libraries were sequenced on a NextSeq 550 System, subsampled to 30M reads. Percent duplicates were calculated by subsampling to 4M reads and analyzed using the BaseSpace RNA-Seq Alignment App v2.0.Bottom- Performance metrics for Illumina Stranded Total RNA Prep with Ribo-Zero Plus
dat Illumina公司总RNA链准备达到高a concordance between (A) input amounts of 1 ng and 100 ng UHR RNA and (B) technical replicates of 10 ng FFPE RNA. Libraries were sequenced on a NovaSeq 6000 System at 2 × 74 bp. Data analysis was performed using the BaseSpace RNA-Seq Alignment App v2.0.1.
Illumina Stranded Total RNA Prep delivers a fast workflow with reduced handson time. Times may vary depending on equipment used, number of samples processed, automation procedures, or user experience.
Data Sheet | PDF |6 versions
Best practices for read trimming for Illumina Stranded mRNA and Total RNA workflows
Technical Note | PDF< 1 MB
Application Note | PDF< 1 MB
Brochure | PDF1 MB
Illumina library preparation solutions
Brochure | PDF2 MB
Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus Consumables & Equipment Documentation
Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus Documentation
Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus Reference Guide Documentation
Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus Checklist Documentation
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All Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus Support
Data management and simplified bioinformatics for labs getting started and for rapidly scaling next-generation sequencing (NGS) operations.
A simple, scalable, cost-effective, rapid single-day solution for analyzing the coding transcriptome leveraging as little as 25 ng input of standard (non-degraded) RNA.
Provides a reproducible, economical solution for sequencing RNA from FFPE tissues and other low-quality samples. Accuracy from as little as 10 ng total RNA.
Achieve rapid, targeted interrogation of an expansive number of target genes with exceptional capture efficiency and coverage uniformity.
A fast, integrated workflow for a wide range of applications, from human whole-genome sequencing to amplicons, plasmids, and microbial species.
Prepare sequencing libraries for small genomes, PCR amplicons, plasmids, or cDNA in as little as 90 minutes, with a low DNA input requirement.