The Ion Total RNA-Seq Kit for AB Library Builder™ System combines optimized reagents and protocols for automated preparation of representative cDNA libraries for strand-specific RNA sequencing of all types of RNA species on the Ion Personal Genome Machine (PGM™) and Proton™ sequencers. Either small RNA (such as microRNA) or whole transcriptome RNA samples can be prepared for next-generation sequencing.
The Ion Total RNA-Seq Kit for AB Library Builder™ System contains the same performance advantages as the manual Ion Total RNA-Seq Kit v2
with the convenience of protocol automation. The gel size selection step in the small RNA workflow and the filter cleanup steps have all been replaced with a magnetic bead-based method that reduces the total reaction time to approximately 6 hours. This automated workflow eliminates the hassles associated with manually producing RNA libraries for RNA expression analysis applications. This instrument and reagent combination provides researchers the ability to produce ~36 libraries in a typical work day for medium to high throughput sequencing labs.
The Ion Total RNA-Seq Kit for AB Library Builder™ System is designed to make cDNA library preparation for semiconductor sequencers simple, fast, and flexible. It can be used to generate a representative cDNA library from any type of RNA sample. The Ion Total RNA-Seq Kit for AB Library Builder™ System provides an automated total RNA library solution employing a common workflow for the discovery of small RNAs and isoforms, coding RNA, noncoding RNA, and alternative splice variants.
Additional Features of the New Ion Total RNA-Seq Kit for AB Library Builder™ System:
• Automated workflow—magnetic bead-based purification simplifies automation of library steps post RNA fragmentation
• Greater accuracy—SuperScript® III 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
As with the manual Ion Total RNA-Seq Kit v2, the Ion Total RNA-Seq for AB Library Builder™ System:
• 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) RNASmall 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 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 for AB Library Builder™ System 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 to approximately 3 hours on the instrument. A separate Magnetic Bead Cleanup Module is included with the kit and additional modules may be purchased separately if needed.
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 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.