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Ion Total RNA-Seq Kit v2

Catalog number:  4475936

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The Ion Total RNA-Seq Kit v2 includes the reagents needed to prepare representative cDNA libraries for strand-specific RNA sequencing on the Ion GeneStudio™ S5, Ion Proton™, or the Ion Personal Genome Machine™ (PGM™) systems. The Ion GeneStudio S5 and Ion Proton systems are ideally suited for sequencing the whole transcriptome (ribosomal RNA depleted or polyA), while the Ion GeneStudio S5 and Ion PGM systems are suitable for small RNA, as well as viral and bacterial transcriptomes. Either small RNA (such as microRNA) or whole transcriptome RNA samples can be prepared for next-generation sequencing using the Ion Total RNA-Seq Kit v2. Version 2 of the Ion Total RNA-Seq Kit is an improvement over the first generation kit. The gel size selection step in the small RNA workflow has been replaced with a magnetic bead-based method. Magnetic bead-based purification replaces all of the filter cleanup steps and the total reaction time has been reduced to 6 hours.

Additional Features of the New Ion Total RNA-Seq Kit v2:

• Greater accuracy—SuperScript® VILO™ and Platinum® PCR SuperMix High Fidelity added for highest template fidelity
• Barcode compatible—works with Ion Xpress™ RNA-Seq Barcode 01-16 Kit for multiplexing
• Automation friendly—magnetic bead-based purification simplifies automation of library construction

As with the previous kit, the Ion Total RNA-Seq Kit v2:

• Preserves strand information—all mapped reads are aligned in the direction of transcription relative to the chromosomal strand
• Allows you to choose your workflow—interrogate either small RNA or the whole transcriptome
• Allows you to analyze any type of RNA—supports small RNA, rRNA depleted total RNA, and poly(A) RNA

The Ion Total RNA-Seq Kit v2 is designed to make cDNA library preparation for the Ion Proton™ and PGM™ systems fast and flexible. It can be used to generate a representative cDNA library, flanked by specific sequences necessary for sequencing, from any type of RNA sample.

Designed as a complete solution with a common workflow, the Ion Total RNA-Seq Kit v2 combines optimized reagents and protocols for discovery of small RNAs and isoforms, coding RNA, noncoding RNA, and alternative splice variants.

Small RNA Analysis
During small RNA library construction, the 3' and 5' adaptors are attached directionally and simultaneously. As a by-product of this step, an adaptor:adaptor product may be formed without an RNA insert. This byproduct will amplify during first strand synthesis and PCR. If not removed, > 50% of the reads will be the adaptor dimer. Historically, the only way to separate the adaptor dimer from the wanted small RNA containing library fragments has been through gel size selection. The Ion Total RNA-Seq Kit v2 uses proprietary technology to inhibit cDNA synthesis of the adaptor byproduct, thus allowing cDNA separation with magnetic bead-based technologies. Total reaction time has been reduced from 1.5 days to approximately 6 hours. A separate Magnetic Bead Cleanup Module is included with the kit. Additional modules may be purchased separately.

Start with total RNA containing as little as 5–100 ng of miRNA, or RNA enriched for small RNA containing 1–100 ng of miRNA. The small RNA protocol provides guidance on whether to start with total RNA or RNA enriched for small RNA, based on the small RNA content of your sample. Small RNA enrichment protocols are also provided.

Whole Transcriptome Analysis
The whole transcriptome protocol enables construction of strand-specific libraries in approximately 5 hours. Starting with as little as 100 ng of total RNA, construct a library from 1 ng of poly(A) RNA or 25 ng of rRNA-depleted RNA following the RNA enrichment and library generation protocols provided in the manual. Because the libraries are not limited to cDNA derived only from poly(A) RNA, Ion Total RNA-Seq Kit libraries support a more thorough investigation of transcriptome complexity, capable of characterizing known and undocumented transcripts, including alternative splice variants, fusion transcripts, and SNPs.

Preserve Strand Information
Unlike methods that ligate adapters to double-stranded cDNA, the Ion Total RNA-Seq Kit v2 utilizes proprietary Ambion® technology to attach the adapters in a directional manner that preserves strand information in the resulting libraries. In addition, both the 3' and 5' adapters are attached simultaneously, reducing ligation and clean-up steps.

Preserving strand orientation during library construction helps enable more accurate determination of the structure and expression level of transcripts, and can aid in the discovery of novel transcription regions from both the positive and negative genomic strands.

The Ion Total RNA-Seq Kit v2 is designed to create RNA libraries from up to 48 samples for small RNA or whole transcriptome sequencing on the Ion Proton™ or PGM™ systems.
For Research Use Only. Not for use in diagnostic procedures.


For Use With (Equipment): Ion PGM™ System
Workflow Step: Library Generation

Contents & storage

Ion RNA-Seq Core Kit v2

Store at -20°C to room temperature:

2 x 1.75 ml Nuclease-free Water

Store at -20°C:

1 x 20 µL 10X RNase III Reaction Buffer

1 x 20 µL RNase III

1 x 40 µL Hybridization Solution

1 x 150 µL 2X Ligation Buffer

1 x 30 µL Ligation Enzyme Mix

1 x 30 µL 2.5 mM dNTP Mix

1 x 56 µL 10X SuperScript® III Enzyme Mix

1 x 900 µL Platinum® PCR SuperMix High Fidelity

1 x 50 µL WT Control RNA (1 µg/µL HeLa total RNA)

1 x 10 µL Small RNA Control (1 µg/µL human placenta total RNA)

1 x 10 µL Small RNA Control (1 µg/µL human placenta total RNA)

Ion RNA-Seq Primer Set v2 (Cat. No. 4474810)

Store at -20°C:

1 x 30 µL Ion Adaptor Mix v2

1 x 104 µL Ion RT Primer v2

1 x 20 µL Ion 5' PCR Primer v2

1 x 20 µL Ion 3' PCR Primer v2