I want to make a library for RNA-seq from a total RNA extracted from a complex system that includes eukaryotes (plant and fungi) and prokaryotes (bacteria). Does anyone have clues on how to perform rRNA depletion form such a sample? I have read a manuscript in which they use the Ribominus eukaryote kit adding yeast and prokaryote probes (PMC5081961), any comment on that?
Helo
Yes, I've done depletion of rRNA from mixed sample plant/bacteria (plant nodules with rhizobia) with kit RiboZero and got very good results in RNAseq. I've mixed two types of probes (plant and bacteria) to go through only one step of depletion. To check the compatibility of the probes with your species you can ask directly the producer.
Please take a look at this discussion (on RG). It might offer you some insights and possible problems you might encounter in your experiment. It was posted about 3 years ago. By now, probably there are better commercial kits or techniques for this kind of experiments.
https://www.researchgate.net/post/Experience_with_double_rRNA_depletion
Thanks Agnieszka and Yuan-Yeu,
Agnieszka, did you use equal amounts of the two probes? I am afraid that the high abundance of plant RNA (my sample is a rhizosphere) may outnumber the plant probes and leave a lot of plant rRNA reamining.
Thanks so much again!
It depends on the biological material you have to adjust the proportion of probes. And also you system is more complicated as you have three organisms.
I've used the equal amounts of two probes. However, I've used much less of total RNA per run of depletion than advised by the protocol (5µg instead of max which is 10 µg) to keep the probes in excess. Consequently, I had to do 3 depletions in parallel to get enough material for RNAseq.
If you have sufficient quantities of samples, make two runs of depletion (or make first depletion of plant rRNA, and then plant and bacteria rRNA). Advise: after each step of depletion analyze your RNA with Bioanalyser to follow the progression of rRNA depletion. I hope it helps.
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