RNA Sequencing (RNA-seq)

What is RNA-seq?

RNA-sequencing (RNA-seq) is a powerful method for studying the transcriptome—the entire spectrum of coding and noncoding RNA transcripts present in a cell or tissue. The transcriptome can provide valuable insights into cellular behavior at a particular moment, whether at rest or when responding to changes in the environment, therapeutic treatments, or developmental progression. RNA-seq also enables the identification of variant splicing events, novel transcripts, mutations, gene fusions, isoforms, and single nucleotide variants (SNVs).

RNA-seq strategies and workflows

The best RNA-seq workflow for your experiment depends on the questions you are trying to answer, the quality of the RNA sample, the amount of RNA available, and the nature of the transcripts of interest. The KAPA RNA HyperPrep Kit is a robust, streamlined library preparation solution for the preparation of total RNA libraries, and forms the basis for KAPA RNA-seq workflows for transcriptome sequencing with ribodepletion, custom depletion, and mRNA capture workflows (see below).

Depletion of rRNA, user-defined RNA, and globin RNA

Whole-transcriptome sequencing provides a complete view of the transcriptome, including immature and noncoding RNAs. In these workflows, it is important to remove rRNA (up to 95% of the total RNA sample) prior to library prep in order to minimize the number of wasted sequencing reads. This method is effective with degraded RNA inputs not suitable for mRNA capture. Enzymatic depletion can also be used to remove other transcripts, such globin mRNA and/or other highly abundant RNAs.

mRNA capture

mRNA capture sequencing is used for the analysis of coding transcripts in eukaryotic samples, allowing researchers to detect and quantify relative expression levels. This workflow enriches for mRNA over non-polyadenylated species such as ribosomal, precursor, and non-coding RNAs.

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Figure 1. Overview of workflows for rRNA depletion, globin, or custom depletion and for mRNA enrichment using KAPA RNA HyperPrep Kits. The KAPA RiboErase workflow is the first part of the KAPA RNA HyperPrep Kit with RiboErase Kit (HMR) workflow; the oligos can be replaced with custom oligos. The mRNA depletion workflow is the first part of the KAPA mRNA HyperPrep Kit workflow. Both kits leverage the single-tube library preparation workflow of KAPA RNA HyperPrep. (See the figure for workflow details.)

RNA-seq library prep is the critical first step

Roche’s KAPA RNA HyperPrep kits offers flexible workflow options with streamlined single-tube single-day workflows that include enrichment of desired transcripts by selective mRNA capture or rRNA depletion. These kits have been optimized for high performance even with degraded and low-input samples, and offer the option of custom RNA depletion.

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Figure 2. KAPA RNA HyperPrep provides streamlined, strand-specific library construction. The novel chemistry employed in KAPA RNA HyperPrep Kits allows for fewer and shorter enzymatic steps, reducing hands-on time and overall library prep time. rRNA depletion with KAPA RiboErase (HMR) or KAPA RiboErase (HMR) Globin Kits adds approximately 2.5 hours to the overall workflow time, whereas mRNA capture adds approximately 1.5 hours. The entire workflow, from input RNA to sequencing-ready library, can easily be completed in a standard workday. All KAPA RNA HyperPrep library construction workflows are automation friendly.

Automate RNA-seq library preparation

Automation increases throughput, reduces hands-on time, reduces human error, and enables workflow standardization. It also minimizes potential pipetting errors and standardizes workflows for improved results. Validated scripts are available for KAPA RNA HyperPrep Kits for many liquid handling instruments.

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Streamline RNA-seq data analysis for KAPA RNA HyperPrep Kits – no bioinformatics experience required

Managing the enormous amount of RNA-seq data is a challenge, and it can take weeks to receive an analysis report from external bioinformaticians. Roche collaborates with Genialis to offer a biologist-friendly cloud-based analysis application that runs raw sequencing data through preconfigured automated pipelines, tracks how each sample was processed, visualizes and interprets results in real time, and manages all the projects in the cloud.

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