Removing rRNAs

First shared at my LinkedIn page: https://www.linkedin.com/posts/activity-6752205954168823808-cuDf

As you might know, total RNA is dominated (~80%) by ribosomal RNAs (rRNA). mRNA is only ~5% of it. There is also so called non-coding RNAs as sub-categories like small < 30nt (i.e. miRNAs), and long >200nt RNAs in it. Ribosomal RNAs are not the main interest of the most transcriptomic studies. Morever, the one who carries the “information” (historically, in this case this refers to close relationship with the “doers” of a cell, proteins, so I call it as “translationability”) are mRNAs.

However, non-coding RNAs have many regulatory function. Let’s say you want to sequence mRNA or these non-coding precious “junk”(matter of “to be or not be” proteins). In this case, we need to make sure that there is no/minimum rRNA around to interfere with our precious and limited amount of messenger and modulator or mysterious (=with unknown role) RNAs for the sequencing. Although, mRNA enrichment (i.e. capturing from polyA tails) like methods are also available, sometimes you need to have more options.

To best of “my” knowledge, there are 3 main methods available for rRNA depletion, all of them are targeting specific sequences (=rRNA): pulling with beads, oligos following RNAseH digestion and with CRISPR/Cas9 (yep, 2020NobelPrizeWinner is here as well).

If you want to learn more about the efficiency of these and understand the similarities and differences, there are couple of articles available. I would like to share those for the researchers interested in:

• Ribo-Zero Gold Kit: improved RNA-seq results after removal of cytoplasmic and mitochondrial ribosomal RNA (Nat. Met., 2011)
• Comparative analysis of RNA sequencing methods for degraded or low-input samples (Nature, 2013)
• Depletion of Abundant Sequences by Hybridization (DASH): using Cas9 to remove unwanted high-abundance species in sequencing libraries and molecular counting applications (Genome Biology, 2016)
• Comparison of Ribosomal RNA Removal Methods for Transcriptome Sequencing Workflows in Teleost Fish (Animal biotechnology, 2016)
• Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction (BMC Genomics, 2018)
• Evaluation of protocols for rRNA depletion-based RNA sequencing of nanogram inputs of mammalian total RNA (PlosONE, 2019)
• Scalable and cost-effective ribonuclease-based rRNA depletion for transcriptomics (Bacterial work) (NAR, 2019)
• A Simple, Cost-Effective, and Robust Method for rRNA Depletion in RNA-Sequencing Studies (ASM, 2020)