Qiagen, 331885, QIAseq UX 96 Index Kit IL UDI-H (96)

Sample Index Kit Set H for 96 libraries using unique dual indices on Illumina NGS instruments; indices are supplied as liquid in a 96 well plate with a pierceable foil seal

Features

- Start with 1–1000 ng of total RNA or enriched mRNA
- Integrated QIAseq FastSelect RNA removal reagent suppresses ribosomal RNA or other unwanted RNAs
- Sample barcoding following reverse transcription ensures no sample mix-ups
- Includes protocols for library construction for whole transcriptome or 3’ RNA-seq starting with total RNA or enriched mRNA
- Includes access to online RNA-seq read mapping, differential gene expression and pathway analysis using the RNA-seq Analysis Portal for supported species

Product Details

QIAseq FastSelect RNA Library Kits utilize a simple and fast workflow for complete transcriptomics starting from 1 ng of total RNA. The simple workflow utilizes the FastSelect RNA removal reagents to suppress ribosomal RNA and other unwanted RNAs to enable gene expression and transcript identification from a variety of model systems including human, mouse, rat and related species, plants, yeast, zebrafish, flies, worms and bacteria. Researchers can also utilize mRNA enrichment kits (sold separately) and focus RNA-seq library construction on protein coding mRNAs. When used with the QIAseq UX Index IL UDI Index Kits (sold separately), 768 individual samples can be combined together for a single sequencing flow cell lane.

The complete solution includes online access to the RNA-seq analysis portal (for supported species), which allows researchers to start with FASTQ files and perform read alignment, differential gene expression and pathway analysis. Furthermore, qPCR and digital PCR assays for biological verification can be easily identified in QIAGEN GeneGlobe following data analysis. QIAseq FastSelect RNA Library Kits are also supported with on-site software through QIAGEN’s CLC Genomics Workbench (sold separately).

The final RNA-seq libraries created using the QIAseq FastSelect RNA Library Kits retain strand and directionality of the original transcript, increasing the accuracy of the final mapped transcripts and quantification.

Performance

QIAseq FastSelect RNA Library Kits are versatile and contain all the reagents to make stranded RNA-seq libraries for complete transcriptomics or 3’ RNA-seq, depending on the workflow selected and type of RNA used. This provides researchers with the ability to choose the appropriate library type based on the input RNA, NGS instrument, read budget and overall project goals. RNA-seq libraries have been successfully constructed from high-quality total RNA, fragmented FFPE RNA and enriched mRNA. To see demo data, log in to the RNA-seq Analysis Portal and go to the QIAseq FastSelect RNA Library folder.

Principle

QIAseq FastSelect RNA Library Kits have several innovative advantages compared to other RNA library kits:

First, the integration of QIAseq FastSelect in the workflow allows for rapid and efficient removal of ribosomal RNA during the cDNA synthesis reaction. In one step, QIAseq FastSelect removes up to 99% of all unwanted rRNA, even when starting with difficult samples or instances where the RNA is already degraded, such as formalin-fixed paraffin embedded (FFPE) samples.

Second, during reverse transcription, each transcript incorporates a unique sample ID. Following the reverse transcription step samples can be tracked to prevent any sample mix-ups. During sample indexing and final library amplification, up to 768 different unique dual indices can be utilized.

An alternative protocol is provided that allows for cDNA pooling following the reverse transcription step. When utilizing cDNA pooling, more than 10,000 samples can sequenced together in a single flow cell lane.

Procedure

Total RNA or enriched mRNA is heated and then stepwise cooled to allow QIAseq FastSelect to block ribosomal and unwanted RNAs. Next, the treated RNA is reverse transcribed into cDNA. Researchers choose which reverse transcription primers to use for building their RNA-seq libraries: random hexamers, oligo-dt or combine both primers for complete RNA coverage. During the reverse transcription process, each sample receives a unique sample ID to avoid sample mix-ups.

Following cDNA synthesis, each library is amplified and unique dual indices are added using the QIAseq UX Index Kits in a single PCR step. Libraries are quantitated for yield and fragment size, pooled and then sequenced on an Illumina NGS instrument. An optional pooling step after cDNA synthesis can be utilized to simplify library construction and increase the sample number per sequencing run.

The complete workflow from total RNA to sequencing-ready library usually takes less than 5 hours of total time, with only 2.5 hours of hands-on time.

For RNA-seq data analysis, FASTQ files can be uploaded to QIAGEN GeneGlobe for use with the RNA-seq Analyis Portal for sample demultiplexing, differential gene expression and pathway analysis. This online tool contains commonly used NGS pipelines and algorithms specifically designed for QIAseq FastSelect libraries. Simply upload or transfer your FASTQ files and configure the experiments without downloading or investing in any computer harware or software.

Applications

QIAseq FastSelect RNA Library Kits support stranded RNA-seq library construction for transcript identification, fusion gene discovery and high-throughput gene expression. Input RNA can be high quality or fragmented, allowing for RNA-seq libraries from tissues, exosomes, FFPE samples and cell lines. RNA from eukaryotic, prokaryotic and fungi samples is also supported.