Service summary

• RNA extractions with QIAsymphony Paxgene Blood RNA Kit on QIAsymphony*
• QC check with Invitrogen Qubit, Agilent Bioanalyzer and Tapestation
• Library preparation and sample indexing with Illumina kits
• Full automated library preparation on Hamilton NGS STAR
• Sequencing with NovaSeq 6000, ≥25 million reads (R1 + R2 = 50 M Reads) output per sample
• Turnover time approx. 4 to 5 weeks**

(*) For unextracted samples.
(**) Turnover times are not guaranteed, may change according to your project.

Sample requirements

Accepted Sample Type	Recommended Amount	Shipment method
Whole Blood (Fresh/Frozen)	2.5 mL in PaxGene tube 650μL -1mL in 2mL tubes	RT for ≤ 3 days Dry ice for ≥ 3 days
Tissue	30 - 50 mg	Dry ice
FFPE Tissue	4-6 slices for 10µm, 8-10 slices for 5µm	Room temprature
Cell Pellet	≥10^6 cells (eukaryotic) ≥10^8 cells (microorganism)	Dry ice
Total RNA	50μl, >30 ng/μL RIN ≥7.0	Dry ice

How to evaluate the sample quality

We check 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). 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. Do not use absorbance measurements (Nanodrop, spectrophotometer).

RNA quality

Capillary electrophoresis (Fragment Analyzer, Bioanalyzer, TapeStation), RIN ≥7.0. 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 SZAOMICS, 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, SZAOMICS 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

At SZAOMICS, we offer whole transcriptome sequencing using the Illumina Stranded total RNA Prep (Ribo-Zero Plus) Kit.

The total-RNA samples are depleted from abundant transcripts, such as ribosomal RNA, using Ribo-Zero 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

Illumina’s 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.

We evaluate the concentration of the libraries using qPCR and look at the fragment size distribution of the library molecules using Bioanalyzer. Once the libraries have passed this QC step, they are normalized, pooled, and queued for sequencing.