RNA-seq of human cells and bacteria

Question

[bart]

Hey,

I want to study bacterial RNA which may be present inside human cells. The transcriptome of the human cells have been studied using RNA-sequencing in a past experiment. However, in that past experiment, any possible presence of bacterial RNA has not been studied. The RNA-sequencing experiment used the SMARTer protocol (http://www.bea.ki.se/documents/SMART_v4_User%20Manual.pdf) to create cDNA from mRNA. In this protocol, a primer binds complementary to the (human) mRNA poly-A tail to eventually create cDNA from the human mRNA.

I have read that bacteria don't have a poly-A tails and therefore, analysis of bacterial RNA would not be possible in theory, as cDNA from bacteria cannot be created. Does anyone know if this is true or does anyone have some suggestion to study bacterial RNA in human cells?

Kind regards, Bart

[ATpoint]

Yes, bacteria do not polyadenylate their transcripts. You would need to extract mRNA via a workflow that does not use polyA capture, and deplete ribosomal RNA via a ribo depletion procedure, and finally make cDNA with random hexamer rather than a poly-dT primer. This is mostly wetlab and not bioinformatics, but for all that kits exist. I'd just google for bacterial RNA-seq library preparation (or ask a sequencing facility for a quote), probably you would then apply this workflow to total RNA from your human cells. There are probably better communities for this, e.g. a NGS/wetlab-focused subReddit.

[bart]

Thanks for your response. I want to study any RNA molecule other than mRNAs created in the aforementioned experiment. Do you know which RNA molecules apart from mRNAs have a poly-A tail and could still be studied? From what I can find only some lncRNAs may have a poly A tail

[ATpoint]

I think at this point you should consult with experienced folks and your PI in a face-to-face fashion to properly plan and discuss your experiment, finding out which questions you want to answer and which RNA types you even want to include. I doubt that biostars is the proper place to discuss and plan the very basics of an NGS experiment. Be sure to get a proper background first in RNA biology first as you seem right now to be rather guessing about the experimental plans you have (no offense intended). Data analysis can do many things but a proper experimental design is the foundation for everything.

Answer 1

[Mensur Dlakic]

You will not be able to use a kit that is based on poly-A tails, because bacterial transcripts do not have them. As ATpoint suggested, you can use random priming for bacterial transcripts, but this would still give you a bunch of human transcripts. I suggest you first pass the cells through a filter that will allow only bacteria to go through. This may be of interest:

https://www.nature.com/articles/s41378-019-0063-4

[bart]

Thanks for your response. I want to study any RNA molecule other than mRNAs created in the aforementioned experiment. Do you know which RNA molecules apart from mRNAs have a poly-A tail and could still be studied? From what I can find only some lncRNAs may have a poly A tail