RNA sequencing

Bulk RNA sequencing is a 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 methods available for preparing samples for RNA sequencing. Below are some methods that are available at BEA:

  • Whole transcriptome sequencing or total RNA sequencing makes it possible to detect novel splice variants and analysis of both coding and non-coding RNA expression. Generally, rRNA are first depleted in the library preparation process.
  • 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.
  • Targeted RNA sequencing focus on specific transcripts of interest. Different selected library preparations by PCR or hybridization capture are available.

Different library preparation methods have different requirement for RNA input concentration and quality. Degraded input material will generate low complexity libraries and the number of unique fragments in the sequencing library will be low which will affect the outcome of the analysis. Choosing the right library preparation method is therefore crucial for the analysis outcome.

Choosing the right library preparation method is therefore 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 Stranded Ligation assays offers a streamlined, cost efficient, and scalable solution for transcriptome analysis. It is compatible with a wide range of RNA sequencing samples and targets different pools of RNA. Alternatively, the NEB ultra II RNA Kit can also be used with similar results. Important considerations can be found in the protocols under the links below.

mRNA-seqILLUMINAIllumina stranded ligation mRNA25-1000 ng total RNA≤25ul
Total RNA-seqILLUMINAIllumina stranded ligation Total RNA with rRNA depletion10-1000 ng total RNA≤25ul
RNA-seqNEBNEBNext Ultra RNA Library Prep 10-1000 ng total RNA≤20ul
SMART-seqTAKARATakara SMART seq0,5-10 ng Total RNA≤5ul

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 and single cells we use the SMART-Seq reagents from Takara. More information under the single cell sequencing section.

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.