Jun 28, 2023

RNAseH-based ribodepletion

DOI
https://dx.doi.org/10.17504/protocols.io.36wgqj9n5vk5/v1
  • 1Garvan Institute for Medical Research;
  • 2Centre for Genomic Regulation;
  • 3Center for Genomic Regulation (CRG)
Eva Maria Novoa
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DOI: https://dx.doi.org/10.17504/protocols.io.36wgqj9n5vk5/v1
Protocol Citation: Nicole Schonrock, Helaine Graziele Santos Vieira, Oguzhan Begik, Eva Maria Novoa 2023. RNAseH-based ribodepletion. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqj9n5vk5/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 12, 2022
Last Modified: June 28, 2023
Protocol  Integer ID: 69866
Keywords: using rnaseh nuclease, rnaseh enzyme, rnaseh nuclease, based ribodepletion goal, rnaseh, ribodepletion goal, rna sample, rna molecule, dna oligonucleotide, complementary to human rrna, rna, approaches as nuclease, dna oligo, samples from rrna, rrna molecule, ribodepletion, human rrna, many rounds of ribodepletion, ribodepleted material, nuclease, rrna, 28s ribodepletion, dna, enzyme, dna duplex
Disclaimer
Do not use for long read approaches.
This protocol is adapted from the protocol used in Adiconis et al., Nature Methods 2013 (https://doi.org/10.1038/nmeth.2483)
Abstract
Goal: Method to ribodeplete samples from rRNAs using RNAseH nuclease.
Summary: DNA oligos are complementary to human rRNAs. They will anneal to the rRNA molecules. RNAseH will degrade RNA-DNA duplexes, leaving the RNA samples depleted from rRNAs
Tested on :Human, Mouse and Yeast (for 18s and 28s ribodepletion).
Cost: DNA oligonucleotides will be expensive the first time ordered, but will last for many rounds of ribodepletion. Once the DNA oligonucleotides have been purchased, the major cost comes from the RNAseH enzyme. In our hands, the cost is much lower than commercial kits, thus being scalable for high input samples where the ribodepleted material needs to be high.
Recommended for: NGS-based approaches. Do not use for Nanopore-based approaches as nuclease-based methods degrade RNA molecules.