Ribosomal RNA (rRNA) can get in the way, so here are some methods to take it away! Ribosomes (the protein-making machinery in cells) are made up of proteins and rRNA. Cells have a lot of ribosomes (because they need to make a lot of proteins). So cells have a lot of rRNA. Like, a lot a lot (~90% of cellular RNA). And, when it comes to creating sequencing libraries, want that rRNA you do not!

There are a couple of main methods to remove it that rely on using DNA probes complementary to rRNA sequences. These probes bind to the rRNA and allow you to selectively degrade it (e.g. with RNaseH that cuts RNA/DNA duplexes) or physically remove it (e.g. with biotinylated probes you can get to stick to magnetic beads).

I’m going to be using that second strategy, the subtractive hybridization, when I do my ribosome profiling experiments, where I want to see what sequences ribosomes are bound to – not what sequences make up the ribosomes! More on ribosome profiling (aka ribosome footprinting aka RiboSeq) here: https://bit.ly/ribosomefootprinting 

When doing it, there are some special considerations that make it so the nuclease-mediated depletion methods are less than ideal (they can cause bias and inaccuracy when it comes to exact ribosome positions). More on that here:

Zinshteyn B, Wangen JR, Hua B, Green R. Nuclease-mediated depletion biases in ribosome footprint profiling libraries. RNA. 2020 Oct;26(10):1481-1488. doi: 10.1261/rna.075523.120. Epub 2020 Jun 5. PMID: 32503920; PMCID: PMC7491325. https://doi.org/10.1261/rna.075523.120 

So it’s recommended you use the subtractive hybridization method instead. I’m using depletion probes based on this paper:

Ingolia, N., Brar, G., Rouskin, S. et al. The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nat Protoc 7, 1534–1550 (2012). https://doi.org/10.1038/nprot.2012.086 

And this paper talks about a software program you can use to design optimal custom probes for your experiment to maximize the rRNA minimizing!

Ferhat Alkan, Joana Silva, Eric Pintó Barberà, William J. Faller, Ribo-ODDR: oligo design pipeline for experiment-specific rRNA depletion in Ribo-seq, Bioinformatics, Volume 37, Issue 17, 1 September 2021, Pages 2659–2667, https://doi.org/10.1093/bioinformatics/btab171 

Here’s a nice website with info on different methods as well – https://www.lexogen.com/rna-lexicon-rna-pretreatment-enrichment-or-depletion/ 

And IDT has some nice info about biotinylation – https://www.idtdna.com/pages/education/decoded/article/which-biotin-modification-to-use- & https://www.idtdna.com/pages/education/decoded/article/how-biotin-became-a-tool-of-molecular-biologists 

That’s all for now. Sorry the text is brief, but all these papers I’m reading aren’t! So it’s back to work for me. But hope this helps someone other than just me :). 

more about all sorts of things:  #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0 or search blog: https://thebumblingbiochemist.com           

      

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