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QIAseq UPX 3’ Transcriptome Kits

For high-throughput 3' transcriptome NGS from ultralow amounts of RNA
  • Start with 1–100 cells or 10 pg to 1 ng of isolated RNA
  • LNA-enhanced chemistry for increased accuracy, specificity and sensitivity
  • UMIs eliminate library amplification bias for accurate gene expression
  • UPX tagging allows 4608–18,432 samples per single sequencing lane    
  • Includes cloud-based read alignment and single-cell or low-input analysis
QIAseq UPX 3' Transcriptome Kits enable high-throughput gene expression analysis from single cells and previously isolated RNA. Each kit is enough for 96 or 384 libraries, with kit format comprising of either a 96-well multi-break plates or 384-well single use plates with reverse transcription primers already lyophilized in the bottom of the plate for ease of use. To build libraries for RNA-seq, simply add 1 to 100 cells into each well or 10 pg to 1 ng of RNA to reconstitute a reverse transcription reaction. After cDNA synthesis, combine each plate into a single tube to complete the library preparation. All reagents necessary for starting with cells (such as lysis reagents) or RNA are included in the kit.
Cat No./ID: 333074
QIAseq UPX 3' Trans. 12-Index (48)
For indexing up to 12 samples per sequencing lane on Illumina NGS instruments; multiple 96- or 384-well plates can be combined as one sample allowing for 12 x 96-well plates (1152 cells/wells) or 12 x 384-well plates (4608 cells/wells) per lane
Cat No./ID: 333075
QIAseq UPX 3' Trans. 48-Index (192)
For indexing up to 48 samples per sequencing lane on Illumina NGS instruments; multiple 96- or 384-well plates can be combined as one sample allowing for 48 x 96-well plates (4608 cells/wells) or 48 x 384-well plates (18,432 cells/wells) per lane
Cat No./ID: 333088
QIAseq UPX 3' Transcriptome Kit (96)
For 3' gene expression analysis from cells or mRNA of 8–96 samples at one time; each kit contains cell lysis solution, buffers, oligos and enzymes lyophilized in a 96-well plate which can be easily separated for processing 8–96 samples in parallel (this format is ideal for both small and large sample sizes)
Cat No./ID: 333089
QIAseq UPX 3' Transcriptome Kit (96-M)
For 3' gene expression analysis from cells or mRNA of up to 384 samples; each kit contains cell lysis solution, buffers, oligos and enzymes in a 96-well plate. The increased volumes allow for automation or manual pipetting or where custom protocols are necessary.
Cat No./ID: 333090
QIAseq UPX 3' Transcriptome Kit (384)
For 3' gene expression analysis from cells or mRNA of 384 samples simultaneously; each kit contains cell lysis solution, buffers, oligos and enzymes lyophilized on a 384-well plate for processing 384 samples in parallel (this format is best suited for large samples numbers of at least 384 samples)

QIAseq UPX 3’ Transcriptome Kits适用于分子生物学应用。该产品不适用于疾病的诊断、预防或治疗。


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Library QC and Quantification.
A portion of the 11 μl sequencing library was used as the starting material for the library QC and quantification. Library QC using an Agilent Bioanalyzer is shown with a peak at 425 bp. Real-time PCR-based methods provide an accurate quantification of complete RNA-seq libraries with full adapter sequences. As a result, QIAGEN’s QIAseq Library Quant Array Kit (cat. no. 333304) or Assay Kit (cat. no. 333314), which contains laboratory-verified forward and reverse primers together with a DNA standard, is highly recommended for accurate quantification of the prepared library. Use 1 nM RNA-seq libraries as input for the denaturation procedure to ultimately load 3 pM for the MiSeq (V3 kit) and 1.2 pM for the NextSeq.
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Innovative and Optimized QIAseq UPX 3' Transcriptome Kit Workflow.
Library construction starts with lysis of cells or addition of total RNA to the 96- or 384-well plates that have the reverse transcription primers. Poly-A RNA is reverse transcribed using LNA-enhanced chemistry with an oligo-dt primer containing a random Unique Molecular Index (UMI) and a fixed cell ID. Following reverse transcription, cDNA is combined into a single tube for amplification, fragmentation, end-repair, A-addition and adapter ligation. During library amplification, up to 48 different sample indices can be assigned. The combination of cell IDs and sample IDs enables up to 18,432 libraries to be sequenced simultaneously.
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QIAGEN’s Sample to Insight QIAseq UPX 3' Transcriptome Workflow.
The QIAseq UPX 3' Transcriptome Kit enables high-throughput next-generation sequencing (NGS) of polyadenylated RNAs. The kit is intended for library construction and analysis of single cells, cell pellets and ultra-low amounts of total RNA, starting with single cells, cell pellets or isolated RNA. QIAGEN’s Sample to Insight approach makes transcriptome and targeted gene expression accessible to researchers who demand high-quality results but do not have the time or experience in NGS workflow optimization or have the ability to create complicated bioinformatic pipelines for read alignment and differential gene expression/single-cell analysis.
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Streamlined and Reproducible Workflow Starting from 10 pg to 1 ng of Total RNA.
The QIAseq UPX 3’ Transcriptome Library Kit was used to construct libraries from 10 pg, 100 pg and 1 ng aliquots of HCT 116 total RNA. For each experimental replicate (n=2), reverse transcription and template switching were performed on eight aliquots of each RNA amount. Following this, each set of eight was combined into one tube, since each cDNA is tagged with a unique cell/well ID. Subsequent library construction steps were performed in a single tube and sequencing was performed on a MiSeq. QIAseq UPX 3’ analysis software was used for primary results mapping and deconvolution of the cell/well IDs. The number of captured molecules for each RNA amount are presented, demonstrating the reproducibility of the workflow. 
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Consistency of Detection with QIAseq UPX 3’ Transcriptome Kits.
The QIAseq UPX 3’ Transcriptome Kit was used to construct libraries from 10 pg, 100 pg and 1 ng aliquots of HCT 116 total RNA. For each experimental replicate (n=2), reverse transcription and template switching were performed on eight aliquots of each RNA amount. Following this, each set of eight was combined into one tube, since each cDNA is tagged with a unique cell/well ID. Subsequent library construction steps were performed in a single tube and sequencing was performed on a MiSeq. QIAseq UPX 3’ analysis software was used for primary results mapping and deconvolution of the cell/well IDs. The number of detected genes from each aliquot of RNA (eight per RNA input amount) is presented, demonstrating the consistency of detection.
Performance
The QIAseq UPX 3' Transcriptome Kit presents a unique advantage where each cell is tagged with a unique ID (up to 384 different IDs) and each RNA molecule is tagged with a Unique Molecular Index (UMI) during reverse transcription. Following reverse transcription with integrated template switching, all individually tagged cDNAs can be combined, which enables all subsequent library construction steps to be performed in a single tube – saving significant time and library prep costs. QIAseq UPX 3’ Transcriptome Kits provide a convenient, streamlined and reproducible library construction workflow starting with as little as 10 pg total RNA (see figures "Streamlined and Reproducible Workflow Starting from 10 pg to 1 ng of Total RNA", "Consistency of Detection with QIAseq UPX 3’ Transcriptome Kits and "Library QC and Quantification").
Principle
QIAseq UPX 3' Transcriptome Kits enable Sample to Insight, high-throughput NGS of polyadenylated RNAs from single cells on Illumina NGS instruments. Kits are intended for library construction and analysis of cell pellets (up to 100 cells) and purified RNA (10 pg to 1 ng). QIAseq UPX 3' Transcriptome Kits present an innovative advantage in that during reverse transcription, each cell is tagged with a unique ID (up to 384 different IDs) and each RNA molecule is tagged with a Unique Molecular Index (UMI). Following reverse transcription with integrated template switching, all individually tagged cDNAs can be combined, which enables all subsequent library construction steps to be performed in a single tube. This prevents sample mixup, saves substantial time and dramatically reduces library prep costs. During subsequent amplification and library construction up to 48 different sample IDs can be assigned. Together, the combination of cell IDs and sample IDs enables up to 18,432 libraries to be sequenced together. QIAseq UPX data analysis enables primary mapping, single-cell clustering analysis and differential expression analysis.
Procedure
Innovative, optimized workflow
The QIAseq UPX 3' Transcriptome Kit defines a new generation of high-throughput NGS technologies in QIAGEN’s Sample to Insight workflow. Purified RNA or single cells are first reverse transcribed and each RNA molecule is given a Unique Molecular Index (UMI) and well-specific IDs are assigned (up to 384 wells; cell IDs). Following reverse transcription with integrated template switching, all cDNAs are combined, enabling simplified library construction steps to be performed in a single tube. During subsequent amplification and library construction, up to 48 different library indices (sample IDs) can be assigned. The combination of cell IDs and sample IDs enables up to 18,432 libraries to be sequenced simultaneously (see figures "Innovative and Optimized QIAseq UPX 3' Transcriptome Kit Workflow" and "QIAGEN’s Sample to Insight QIAseq UPX 3' Transcriptome Workflow").

Convenient GeneGlobe data analysis
QIAseq UPX cloud-based data analysis is available via the GeneGlobe Data Analysis Center and provides read alignments, UMI and sample de-multiplexing, with final single-cell or low-input gene expression analysis.
Applications
  • High-throughput screening applications (e.g., toxicology research)
  • Single-cell analysis
  • Differential gene expression analysis