Illumina TruSeq Stranded mRNA
RNA sequencing of mRNAs selected through poly-A enrichment.
Sequencing poly-adenylated messenger RNAs (starting from total RNA), is the standard approach for determination of differentially expressed genes – with a focus on protein-coding transcripts. NGI offers this service using the Illumina TruSeq Stranded mRNA kit.
- Sample type: total RNA
- Concentration: 30-100 ng/μL
- Volume requirement NGI Stockholm: >15 μL (max 100 µL per well). Approx 2 μL of the sample will be used for our initial quality checks, so please account for this when sending us samples.
- Volume requirement NGI Uppsala: 25-50 µl.
Please contact a coordinator if the sample requirements cannot be fulfilled.
- RIN-value: >8
- Amount of material needed for 1 prep: >200 ng (1000 ng recommended)
- Sample extraction method: We recommend using a column-based kit without phenol. The samples should be re-suspended in nuclease-free water.
How to evaluate the sample quality
We check your samples upon arrival, however we still require our users to do their own QC steps before sending samples. To accomplish this we recommend using the following methods.
Fluorometric measurements (Qubit, Quant-it)
Do not use absorbance measurements (Nanodrop, spectrophotometer)
Capillary electrophoresis (Fragment Analyzer, Bioanalyzer, TapeStation). Do not use gel electrophoresis. For FFPE samples we recommend using capillary electrophoresis to determine the DV200 value.
If you are not able to carry out these steps, or your samples are below the required thresholds, please get in touch.
What we do with your samples
Once your samples arrive at NGI, we start by performing a reception control step in which we make sure the sample meets our requirements.
If the samples fail this quality control step, we will contact you to discuss possible options. Should you choose to proceed with samples not fulfilling the criteria it will be at your own risk, NGI will only make one attempt at library preparation and if the prep does not work you will have to pay for the library preparation anyway.
If the samples pass reception control, we will inform you and the samples will be queued for library preparation.
Polyadenylated messenger RNA is captured from the total RNA sample using magnetic beads. After this selection the mRNA is fragmented and cDNA is synthesized. Illumina adapters with sample specific barcode sequences are ligated and the library is amplified using PCR.
Library QC and sequencing
In this step, we evaluate the yield obtained and we will also determine the size distribution of the libraries generated. We will inform you of the QC status of each sample. Once the libraries have passed this QC step, they are normalised, pooled and queued for sequencing.
The sequencing will be carried out following the setup stated in the agreement.
We routinely generate libraries with 200 ng total RNA as input. However, for applications requiring very deep sequencing we recommend using 1000 ng of total RNA.
The sequencing results from each library prep will depend on the characteristics of the sample. For example, if your samples are degraded (low RIN scores) it may be better to choose a library preparation using ribosomal depletion instead, to avoid a 3′ bias of the sequencing reads.