Illumina TruSeq Stranded RNA without selection or depletion

RNA sequencing of either all RNAs in a sample, or of a RNA sample depleted of for example rRNA by the user.

This is an adaptation of the Illumina TruSeq Stranded mRNA protocol where the poly-A selection step is removed and the library is constructed from all available RNA molecules in the sample. Suitable for RNA sequencing of either all RNAs in a sample, or of a RNA sample already depleted of for example rRNA by the user.

Sample requirements

• Sample Type: Purified or amplified mRNA samples or RNA samples for which no purification is required
• Concentration: 10-25 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
• RNA quality: Depends on sample type, if ribosomal depletion has already been done, the RIN score prior to depletion should be >8 or DV200 >20%
• Amount of material needed for 1 prep: >50 ng
• 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.

Concentration
Fluorometric measurements (Qubit, Quant-it)
Do not use absorbance measurements (Nanodrop, spectrophotometer)

RNA quality
Capillary electrophoresis (Fragment Analyzer, Bioanalyzer, TapeStation). Do not use gel electrophoresis. For FFPE samples we recommend using capillary electrophoresis to determine 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.

Library preparation

The RNA samples are chemically fragmented and then turned into cDNA using reverse transcriptase. After a purification to change buffer and remove short fragments the samples are adenylated and illumina index-adapters are ligated. After ligation the samples are purified to change buffer and remove unligated adapters. After this a size selection is performed to remove short and long fragments and narrow the size distribution. After this the samples are amplified and purified before QC.

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.

Last Updated: 9th June 2022