Oxford Nanopore RNA seq

As a Certified Service Provider  in collaboration with DNA Sequencing Facility and Bioinformatics Resource Center the GEC offers long-read nanopore RNA sequencing providing full-length quantification of native RNA or cDNA without fragmentation or amplification bias. Additionally the GEC offers PCR-cDNA preparation in samples with limiting starting material.

updated: February 2019

 

We provide ONT Direct RNA (no barcoding) and cDNA-PCR (with barcoding) library preparation services.

Direct RNA prep requires 1 µg of total RNA or 300 ng of poly(A) tailed RNA in a maximum volume of 8 µl

cDNA-PCR barcoding prep requires 500ng total RNA  in 10 µl

ONT recommendations for RNA quality metrics:

1) RNA samples should be DNase treated

2) RNA concentration should be 150-500ng/µl; if >500ngµl please dilute prior to submission

3) RNA260/A280 ~2.0; A260/A230 ~2.0-2.2

An A260/A280 < 1.8 can indicate the presence of protein or phenol. Additional purification strongly recommended.

If the A260/A230 is significantly lower than 1.8 this is an indiction of the presence of contaminants, and the RNA may need additional purification.

Chemical impurities such as detergents, denaturants, chelating agents and high concentrations of salts should be avoided as these are known to affect the efficiency of enzymatic steps.  Other contaminants such as DNA, proteins and dyes may also reduce the efficiency of steps in the library preparation.

4) RNA should have a RIN ≥ 7 (RNA Integrity Number)

The RIN assigns a measurement of the level of degradation of the RNA in the extract from 1 to 10, with 10 being the least degraded. For more information, go to https://www.agilent.com/cs/library/applications/5989-1165EN.pdf

For service:

  • RNA samples are assayed on the Agilent Tapestation to assess the integrity, NanoDrop to check purity and Qubit for accurate quantification.
  • We request a minimum of 15 µl per sample as 3-4 µl is needed for RNA QC.
  • It is recommended RNA is suspended in nuclease-free water.
  • QC of RNA will occur within 1-3 business days of receipt.  QC results are returned to the client for review.
  • Remaining samples may be returned upon completion of project
  • Sample(s) will be disposed six months upon compleiton of library prep.

Oxford Nanopore Technology Pricing

Service Details UW System Rate External Rate
Direct RNA Service Setup charge for 1-12 concurrent samples

$1,850

$2,877

Direct RNA (SQK-RNA004) Preparation

Per sample charge

$225

$260

cDNA-PCR Service Setup charge for 1-24 concurrent samples

$1,750

$2,722

cDNA-PCR (SQK-PCS114) Preparation Per sample charge $195 $225

Sequencing costs may be found here on the DNA Sequencing Core’s Platforms page.

To request a quote please contact gecinfo@biotech.wisc.edu.

All data and services provided by the University of Wisconsin Biotechnology Center Gene Expression Center are intended for research purposes only. Services are not intended nor certified for diagnostic or clinical use.