Description
The QIAseq Single Cell RNA Library Kit UDI is an end-to-end library preparation solution for RNA-seq from single cells, cell populations or small quantities of RNA. The kit includes all reagents required for cell lysis, reverse transcription, cDNA amplification and PCR-free NGS library preparation, including QIAseq Beads for reaction cleanup and QIAseq Unique Dual-Index Y-Adapter Kits for sample indexing. This kit produces high-quality NGS libraries and generates a tube of excess amplified cDNA that can be stored for follow-up experiments. The kit is highly suited for transcript discovery and differential gene expression from single eukaryotic cells, enriched cell populations and RNA-seq from limited samples, including viral RNA. The QIAseq Single Cell RNA Library Kit UDI allows researchers to focus on mRNAs, explore the transcriptome (mRNAs and lncRNAs) or enrich for small RNA genomes (such as viruses) from low-input RNA samples. This versatile kit enables multiple RNA-seq applications by choosing from several different protocols included with the kit. Analyze RNA-seq data with ease using the GeneGlobe-integrated RNA-seq Analysis Portal – an intuitive, web-based data analysis solution created for biologists and included with this QIAseq Kit.
The kit detects a greater number of transcripts than competing methods at any sequencing depth. In some single-cell RNA-seq workflows, PCR is used extensively for both cDNA amplification and to amplify the minute amounts of library produced. This has a negative effect on library diversity from the loss of low-abundance transcripts due to both stochastic effects and PCR-bias during cDNA amplification and the generation of PCR duplicates during library amplification. Additionally, these PCR systems often introduce GC-bias and length-bias, which can make the sequencing of GC-rich or long transcripts difficult. In contrast, the QIAseq Single Cell RNA Library Kit UDI relies on the REPLI-g enzyme for a highly efficient Multiple Displacement Amplification (MDA) reaction to amplify cDNA. This generates enough material that library amplification is not necessary, delivering a completely PCR-free workflow that eliminates PCR duplicates and maximizes the number of transcripts detected, including long transcripts such as lncRNAs.
This kit relies on three key technologies to enable PCR-free RNA-seq from single cells, populations of cells or low-input RNA. After cell lysis, gDNA degradation and reverse transcription, the cDNA is ligated to generate a long template that can be amplified in an MDA reaction. This is performed using REPLI-g SensiPhi DNA Polymerase, which generates microgram amounts of long cDNA. This long cDNA is then randomly fragmented with QIAGEN’s FX technology into short inserts suitable for sequencing. Finally, a highly optimized and efficient single-tube library preparation packages these inserts into NGS libraries which can be sequenced directly, without the need for library amplification and additional PCR-bias. Taken together, the high-yield MDA, random and unbiased FX fragmentation and efficient single-tube library construction deliver highly complex PCR-free RNA-seq libraries from single cells in only 5.5 hours.
The QIAseq Single Cell RNA Library Kit UDI is a complete cell-to-library solution that generates RNA-seq libraries from single eukaryotic cells or from picogram levels of purified RNA. The kit contains reagents for the efficient lysis of isolated single cells or populations of cells from common cell-sorting or cell-isolation platforms. After lysis, genomic DNA is degraded and reverse transcription is performed. This reaction can be primed using oligo-dT primers to enrich for polyadenylated RNAs, random primers with QIAseq FastSelect for full transcriptome coverage without rRNA, or you can use your own primers to enrich for specific small RNA genomes, like a virus, for sequencing. After reverse transcription and second strand synthesis, a ligation step generates a long, double-stranded cDNA template which is amplified via MDA using the high-fidelity, proofreading REPLI-g SensiPhi DNA Polymerase. This generates microgram amounts of amplified cDNA, some of which can be stored for later use or confirmatory testing. Amplified cDNA is fragmented using QIAGEN’s FX enzymatic fragmentation reaction, generating inserts compatible with common sequencing read lengths. By changing the fragmentation conditions, the insert size can be varied according to individual preferences. The reproducible amplification reaction excludes the need for quantification of the cDNA prior to fragmentation. The fragmented DNA is end-polished and subjected to a highly efficient ligation reaction with the included QIAseq Unique Dual-Index Y-Adapter Kits. Amplification of the library with PCR is not necessary, so the production of PCR duplicates is avoided, and library diversity remains high.
