RNA sequencing

RNA sequencing is highly sensitive and accurate tool for measuring gene expression across the transcriptome. RNA sequencing allows detecting both known and novel features without prior knowledge. There are many different protocols available for preparing samples for RNA sequencing. Below are some methods that are available at BEA:

  • Whole transcriptome sequencing makes it possible to detect novel splice variants and analysis of both coding and non-coding RNA expression.
  • Targeted RNA sequencing focus on specific transcripts of interest. Different selected library preparations by PCR or hybridization capture are available.
  • mRNA sequencing is the most standardized and used method for analyzing gene expression. The method focuses on the coding transcripts and library preparation is performed by initial polyA selection from Total RNA.
  • Small RNA profiling focuses on RNA sequences of <100 nt. Special protocols for capture and library preparation of desired fractions are thus needed.

Choosing the right library preparation method is crucial for the analysis outcome. Total cellular RNA is mainly composed of rRNA and is therefore often not of interest. For the purposes above we offer different library preparation services. The Illumina TruSeq Stranded RNA assays offers a streamlined, cost efficient, and scalable solution for coding transcriptome analysis. It is compatible with a wide range of RNA sequencing samples and targets different pools of RNA. Alternatively, the NEB ultra RNA Kit can also be used with similar results. Important considerations can be found in the protocols under the links below.

For smaller input amounts and pico scale transcriptomics we provide different solutions for library preparation. Input material can be of different origin. Purified Total RNA, cells in lysis buffer or degraded FFPE samples can serve as input material in the analysis. For ultra Low Input we have used the SMART-Seq cDNA syntheiss reagents from Takara or the QIAseq FX RNA reagents from QIAgen.

Targeted RNA sequencing panels can also be prepared with different methods. Predesigned panels with specific genes or gene regions make it possible to increase the sequencing depth in multiple samples allowing the identification of rare variants. Companies like QIAgen, Illumina and Agilent provide solutions for custom designed panels for sequencing on the Illumina platforms. Please inquire for prices and further details.

Additional and very specific RNA sequencing methods are developed continuously. Some methods, like CAGE (Cap Analysis Gene Expression Sequencing) and GRO seq (Global Run-on Sequencing), focus to target transcriptional start sites. A comprehensive selection of different RNA seq methods can be found below. Please inquire for information about any specific library preparation method.

Questions concerning the NGS service at BEA can be answered by Fredrik Fagerstrom-Billai.