Illumina Stranded total RNA Prep (Ribo-Zero Plus)

Total RNA sequencing based on reduced rRNA content and other type of highly abundant RNAs in both prokaryotic and eukaryotic samples.

Illuminas Ribo-Zero Plus depletion kit supports ribosomal RNA (rRNA) depletion from human, mouse, rat, and bacterial species in addition to globin RNAs. The kit is also compatible with low input samples. Total RNA-sequencing with ribosomal depletion offers a comprehensive whole transcriptome analysis by capturing diverse RNA molecules (not small RNA) such as mRNAs, non-coding RNAs that are not polyadenylated and unspliced transcripts.

Sample Requirements

• Sample type: total RNA
• Amount of sample: minimum 300 ng (1000 ng recommended)
• Volume: 25-50μL
• RIN-value: >8
• Amount of material needed for 1 prep: At least 100 ng RNA recommend input. The protocol supports a broad range of RNA inputs, from 1 ng to 1000 ng. The protocol is optimized for 10–100ng RNA input from low-quality RNA (RIN≥2) samples or FFPE (DV200>55%) samples. Library performance can vary with lower input amounts and lesser quality RNA.

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 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, the samples will be queued for library preparation.

Library Preparation

The total-RNA samples are depleted from abundant transcripts, such as ribosomal RNA, using RiboZero Plus depletion reagents. The samples are then chemically fragmented and turned into cDNA using reverse transcriptase, while subsequent ligation and amplification steps add the Illumina index-adapters for clustering and sequencing.

Library QC and Sequencing

We evaluate the concentration of the libraries using qPCR and look at the library fragment size distribution of the library molecules using TapeStation or FragmentAnalyzer. 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: 23rd August 2022