NGI during Covid-19 outbreak

NGI is still up and running during the Covid-19 pandemic, but we are experiencing some limitations in terms of personnel and key reagents. Each NGI node is following its respective host university recommendations and will continue operation until further notice.

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Illumina TruSeq Small RNA

Generation of sequencing libraries from dicer cleaved miRNA.

The TruSeq Small RNA kit is designed to specifically ligate the adapters to the modified 3′ and 5′ end of mature miRNAs. This kit only offers the possibility to pool up to 48 samples using single indexes.

Sample requirements

  • Sample type: total RNA or purified small RNA fragments
  • Concentration: 250-400 ng/μL
  • Volume: >10 μL
  • Amount of material needed for 1 prep: 1000 ng

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
We want you to check that your total RNA extracts contain the small RNA fraction by running capillary electrophoresis using the Bioanalyzer small RNA assay or similar. Do not use gel electrophoresis.

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

First the 3′ and 5′ RNA adapters are ligated to Dicer processed miRNA. Fragments are reverse transcribed, and Illumina adapters containing sample barcodes are added by PCR. An intermediate QC is done before pooling and size selection.

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.

Applications
Bioinformatics Pipelines
Method Status

Service

We are routinely running this method. Please visit the Order Portal to place an order.

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