Illumina TruSeq Stranded total RNA (RiboZero Gold)

RNA sequencing of all RNA in a sample after depletion of rRNA or another type of highly abundant RNA.

Currently Human/Mouse/Rat is the only rRNA depletion kit we keep in stock and users can order any number of samples. Other depletion kits such as globin depletion for blood samples, or plant rRNA depletion can be run but users need to purchase entire kits (eg 48 or 96 reactions). Reactions not used in a project where a whole kit has been purchased cannot be kept for later use. We will do our best to setup projects using the same kits together to save reactions and buy larger kits at reduced prices.

Sample requirements

• Sample type: total RNA
• Concentration: 100-250 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: >500 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.

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 the 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 total-RNA samples are depleted from ribosomal RNA using biotinylated capture probes. These probes are then captured by a magnet and removed. The samples are then chemically fragmented and 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. Lastly, 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: 2nd October 2023