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Old 07-08-2019, 11:58 PM   #1
gretagecse
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Location: Denmark

Join Date: May 2019
Posts: 3
Unhappy no detectable RNA after rRNA depletion

Hi all,

I am working with bacterial RNAseq, and like for many others, the discontinuation of Ribo zero made my life a bit more challenging.

I have been testing an alternative from siTOOLS, called riboPOOLS, but so far I haven't been lucky with it. They are using the oligo method with dynabeads, similar to Ribo zero.

My input RNA is around 2-4 g, within the range that they require. And I follow every step in the protocol, correctly. However, I am having 0 RNA after the depletion. I tried to measure with Quibit HS RNA assay and Bioanalyzer pico chip, and there is nothing left there.

Before depletion, I can see small and mRNAs from the Bioanalyzer results, but they are gone after the depletion.
I heard about very low concentration of mRNA after rRNA depletion, which makes sense due to the high abundance of rRNA, but I am having 0 mRNA or sRNA after the depletion.

I also did a control experiment, where I lowered the concentration of the oligos they suggest, and after that depletion, I had RNA outcome. This convinces me that I am not making a mistake in the protocol.

Has anyone experienced such a thing? Maybe with the Ribo zero before?

Cheers,
Greta
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Old 09-17-2019, 01:12 AM   #2
furor
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Location: Europe

Join Date: Jan 2012
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Hello,

were you able to solve this? I have heard positive news about the pan-prokaryotic kit from the university sequencing center here, and I would be interested in using a combination riboPOOL.

Cheers
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Old 09-17-2019, 07:56 PM   #3
nucacidhunter
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Location: Melbourne

Join Date: Jan 2013
Posts: 1,230
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RiboFree total RNA-seq library prep from Zymo Research looks good on paper and does not require species specific depletion step but I have not tried yet:

https://www.zymoresearch.com/product...na-library-kit
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