REPLI-g Single Cell RNA Library Kit
For RNA library construction from single cells for Illumina sequencing applications
Single cell analysis enables researchers to gain novel insights across a diverse set of applications, including developmental biology, tumor heterogeneity and disease pathogenesis and progression. NGS-based gene expression analyses often require large amounts of cDNA or RNA. Whole transcriptome amplification (WTA) overcomes limited RNA availability by enabling the analysis of a very small number of cells. The REPLI-g Single Cell RNA Library Kit allows reliable investigation of the transcriptome from a single cell with minimal bias. The kit provides unique WTA chemistry in combination with a highly streamlined library construction procedure, optimized to avoid library enrichment and minimize bias associated with PCR amplification. The innovative lysis buffer effectively stabilizes cellular RNA, ensuring that the resulting RNA accurately reflects the in vivo gene expression profile. All enzymatic steps have been developed to enable efficient processing of RNA for accurate amplification of cDNA, which is achieved with negligible sequence bias using innovative Multiple Displacement Amplification (MDA) technology. The REPLI-g Single Cell RNA Library Kit leverages QIAGEN's unique MDA technology and GeneRead library construction technology to prepare a sequencing library with high fidelity and minimal bias, while retaining the sample's unique transcriptional profile.
Streamlined, one-tube library preparation in a single working dayWith the REPLI-g Single Cell RNA Library Kit, reaction setup is straightforward and handling time is greatly reduced, allowing reverse transcription, WTA and library preparation to be completed in a single working day. The kit provides a time-saving, one-tube library preparation protocol that does not require sample cleanup between steps, minimizing starting material loss and cross-contamination risk. Co-optimization of MDA and library construction processes enables a highly streamlined and efficient protocol, reducing MDA time to only 2 hours and eliminating the library amplification step. Optimized enzyme and buffer compositions ensure generation of high-quality, NGS-ready libraries in just one working day.
Minimal bias due to PCR-free workflowIn standard PCR amplification procedures, regions of cDNA with high GC or AT content can result in little or no amplification, leading to misleading sequence data and NGS results. The REPLI-g Single Cell RNA Library Kit employs high-fidelity MDA technology to provide accurate amplification of all transcripts with negligible sequence bias and minimal allelic drop-outs. REPLI-g Single Cell RNA Library Kit contains REPLI-g SensiPhi DNA Polymerase, which, with its proprietary buffer formulation, ensures uniform amplification of cDNA regions that contain highly variable GC content, thereby ensuring even coverage in subsequent sequencing reactions. Costly false-positive or -negative results are minimized with REPLI-g technology due to REPLI-g SensiPhi DNA Polymerase, which has up to 1000-fold higher fidelity compared to normal PCR polymerases. The REPLI-g Single Cell RNA Library Kit combines the advantages of REPLI-g Single Cell technology with the ligation efficiency of GeneRead technology, delivering high-quality libraries ready for NGS, without the need for any library enrichment – avoiding additional amplification bias.
Complete transcriptome coverage, with low experimental variabilityThe REPLI-g Single Cell RNA Library Kit contains novel REPLI-g SensiPhi DNA Polymerase, as well as an optimized set of buffers and reagents for whole transcriptome amplification (WTA) from just single cells, up to 1000 cells, or equivalently small samples. Following efficient cell lysis, complete removal of genomic DNA (gDNA), and sensitive reverse transcription, the kit utilizes Multiple Displacement Amplification (MDA) to uniformly amplify cDNA across the entire transcriptome with negligible sequence bias. Preparing a sequencing library using the REPLI-g Single Cell RNA Library Kit preserves the unique gene expression profile of each individual cell.
Significant number of reads map to protein-coding RNAThe ability to amplify mRNA-enriched RNA (poly A+) makes the REPLI-g Single Cell RNA Library Kit particularly suited to investigate effects on transcription regulation at the single-cell transcriptome level. Amplification of ribosomal RNA (rRNA), which makes up more than 90% of the total cellular RNA population, is virtually eliminated, allowing generation of meaningful mRNA-Seq data. Following the REPLI-g Single Cell RNA Library procedure, >80% of mapped reads belong to protein-coding RNA.
Reliable detection of transcriptsSingle cell analysis can be challenging when transcript abundance varies greatly within a cell. For accurate results, it is essential that whole transcriptome amplification reliably amplifies all transcripts, regardless of their levels within the cell. The sequencing library generated by the REPLI-g Single Cell RNA Library Kit comprises of high numbers of unique transcripts – even from single cells, providing a comprehensive picture of the transcriptome at the single cell level.
Regulation of transcription is driven by a variety of influences, such as stress, cellular environment, or by disease or somatic genomic variation (e.g., point mutations, copy number variations or structural variations). Additionally, transcriptional post-processing, such as alternative splicing, results in a differential transcription pattern and, ultimately, physiology. Because of the composite structure of tissues, investigating transcription regulation in single cells – rather than analyzing a larger number of cells and basing result interpretation on their average behaviour – is of increasing scientific interest.
Following reverse transcription using Quantiscript RT Enzyme Mix, the cDNA is ligated and subjected to WTA. The REPLI-g Single Cell RNA Library Kit uses isothermal genome amplification, termed multiple displacement amplification (MDA), which involves the binding of random hexamers to denatured cDNA. This is followed by strand displacement synthesis at a constant temperature with REPLI-g SensiPhi DNA Polymerase, which has exceptionally strong strand displacement properties. Additional priming events occur on each displaced strand that serve as a template, enabling generation of high yields of amplified cDNA. REPLI-g SensiPhi DNA Polymerase is a DNA polymerase with 3'→5' exonuclease activity (proofreading activity) that delivers up to 1000-fold higher fidelity compared to Taq DNA polymerase. Supported by the unique, optimized REPLI-g Single Cell buffer system, REPLI-g SensiPhi DNA Polymerase easily solves secondary structures such as hairpin loops, thereby preventing slipping, stoppage and dissociation of the polymerase during amplification. This enables the generation of cDNA fragments of up to 100 kb without sequence bias. The REPLI-g Single Cell RNA Library Kit combines the benefits of highly uniform amplification across the entire transcriptome, with negligible sequence bias with fast library preparation — without the need for enrichment, thereby eliminating additional amplification bias.
Unique components of the REPLI-g Single Cell RNA Library Kit
Simple, one-tube procedure – NGS-ready library prep in one day
The REPLI-g Single Cell RNA Library Kit provides a simple and reliable method to efficiently generate RNA libraries suitable for use on Illumina NGS instruments from just a single cell or as little as picograms of RNA in 6.5–7 hours, with only 1.5 hours hands-on time. The kit provides a complete workflow for reliable reverse transcription and highly uniform amplification across the entire transcriptome with negligible sequence bias, followed by fast, one-tube library construction. Dedicated buffers and reagents have been developed to deliver high-quality cDNA from single cells and purified RNA, with complete sequence representation and unbiased amplification.In the first step of the procedure, the cell sample is lysed and the gDNA is removed. Reverse transcription is carried out for 60 min, followed by ligation of cDNAs (30 min). The isothermal amplification reaction then proceeds for 120 min, and can be preprogrammed in a thermal cycler. REPLI-g SC amplified cDNA can be stored long-term at –20°C with no negative effects. Samples consisting of longer cDNA fragments are first sheared into a random library of fragments. The median fragment sizes are dependent on the applications and sequencing read length. Following end-repair, platform-specific adaptors, which contain sequences essential for binding library to a flow cell for sequencing, binding sequencing primer and allowing for PCR enrichment of adapter-ligated cDNA library, are ligated to both ends of the cDNA fragments. The WTA procedure normally results in high yields of cDNA so that library preparation can be performed with a high amount of input cDNA and subsequent library enrichment can be avoided. However, if library enrichment is required, an optional, high-fidelity amplification step can also be performed that provides highly accurate amplification of library cDNA with low error rates and minimum bias.
The REPLI-g Single Cell RNA Library Kit offers an efficient, PCR-free method for RNA library construction from single cells for RNAseq applications on NGS instruments from Illumina. Fields of application include developmental biology, systems biology, characterizing tumor cell heterogeneity and stem cell research.
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