[NicoBxl]

RiboZero is used to rRNA deplete total RNA - there is no size selection involved. Briefly, total RNA is mixed with microspheres & a rRNA removal solution which binds the rRNA and rRNA depleted material is eluted using centrifugation and columns provided in the kit.

So In the sequencing results, there will be also miRNA sequences (and maybe pre-miR sequences ? )

[protist]

So In the sequencing results, there will be also miRNA sequences (and maybe pre-miR sequences ? )

There could be but it would be dependent on how you isolated your total RNA - if you used an isolation method that preserved all the RNAs. Ther is a nice paper discussing rRNA depletion strategies and whole transcriptome analysis:http://www.plosone.org/article/info:...l.pone.0027288. If yourRNA deplete rather than polyA select non coding RNAs will be more likely to be preserved in the subsequent library. However if you are specifically looking for miRNAs an alternate small RNA specific protocol would be the preferred option.

[ETHANol]

As in the main we are still doing 'home brew' strand specific libraries we routinely precipitate our fragmented RNA in the presence of glycogen and have had few problems. Recently for the bacterial libraries we have switched to Zymo RNA (5 ug) Clean&Concentrate columns at the fragmented RNA concentrating stage. We have also used them post the DNase-ing (25 ug variety) for some bacterial RNAs.

Would you mind sharing your protocol as Illumina is completely backordered on their TruSeq RNA kits.

[NicoBxl]

with Ribo-Zero, is the poly-A tail detectable ?

[protist]

For most RNAseq protocols you fragment the RNA and use random hexamers to prime the 1st strand cDNA. The polyA component of your total RNA will still be there after rRNA depletion, the depletion kits only remove small and large subunit rRNA and 5S or 6S RNA and in the case of some kits (RiboZero) the mitochondrial rRNA. What should be left after the depletion is a mix of mRNA (polyA in eukaryotes) and small RNAs.

[NicoBxl]

Ok thanks for all your answers. We will try the Ribo-Zero kit.

[JakobHedegaard]

Contamination with bacterial rRNA

We have done Ribo-Zero Human rRNA depletion of RNA from human clinical samples followed by ScriptSeq library prep and HiSeq sequencing. But it turned out that app 50 % of the reads were of bacterial rRNA origin.
Anyone observed something similar?
/Jakob

[pmiguel]

We have done Ribo-Zero Human rRNA depletion of RNA from human clinical samples followed by ScriptSeq library prep and HiSeq sequencing. But it turned out that app 50 % of the reads were of bacterial rRNA origin.
Anyone observed something similar?
/Jakob

How was the RNA from the human clinical sample isolated? Were reusable centrifuge bottles or tubes used? These are a frequent source of contamination if not scrubbed and then acid washed.

What was your yield of depleted RNA that you processed using ScriptSeq? We have seen a case where we had very limited RNA going into library construction -- far below specifications for the kit. When we found that a low percentage of the reads mapped, we investigated further. Turned out to be bacterial. I am pretty sure it came from the library construction kit.

Also, if this is a human clinical sample is it possible the patient had a bacterial infection?

--
Phillip

[JakobHedegaard]

We are trying hard to locate the source(s) of contamination and have so far been able to amplify 16S rRNA fragments from both DNA and RNA - indicating that the contamination has occured before or during RNA and DNA purification.
We start with 50-500 ng total-RNA and from the bioanalyzer profiles of the depleted RNA we have estimated that ScriptSeq is initiated with "just a few ng's" of RNA - should be fine with ScriptSeq. And we do get excellent human data beside the baterial ones.
Did you observe bacterial contamination when using ScriptSeq? Or was it another library prep kit?
Infection of the patient is of course another option...

Cheers, Jakob

[Olaf Blue]

Contamination?

Jacob, it would be very easy to tell the potential source of the RNA if we have bacterial rRNA sequence information from the sequencing run...if the patient sample IS contaminated with bacteria prior to sequencing, it can certainly be cleaned up with the RZ Kits as I mentioned earlier.

Olaf

[pmiguel]

Did you observe bacterial contamination when using ScriptSeq? Or was it another library prep kit?
Infection of the patient is of course another option...

Cheers, Jakob

No, it was a completely different kit, several years ago. We were using input RNA at least 1 order of magnitude below the specification for the kit. The data was still usable. But I was a little disappointed that it was contaminated.

--
Phillip