Removing rRNA at the DNA stage

[eab]

We need to be able to remove rRNA-derived library molecules from PCR amplified mRNAseq library, rather than removing the rRNA itself during the earlier steps of library construction. We don't need to remove every single rRNA-derived molecule, but would be very happy if we could, say, cut the number of rRNA reads by 75%.

Does anyone know how, other than by DSN?

Thanks!
eab

[SNPsaurus]

Would this work:

http://www.nugen.com/technology/inda-c

or are the libraries already made? Why do you have to remove at the DNA stage?

[eab]

thanks for your reply!
i've heard a bit about that method, not sure how it works exactly, but sadly i do think we need to remove after the libraries are already made.
the reason is i fear there's too little material to attempt to remove ribosomal at the RNA stage. we need to give ourselves a fighting chance with a few extra PCR cycles.

[SNPsaurus]

Our local core director was impressed by the presentation about it at a recent meeting. It does require that the library be made with their adapters, but if you are still at the RNA stage then you could go down that route.

I think the tech is pretty straightforward. Make the library, denature it, throw in a primer to the rRNA region and extend off the template. You'll get dsDNA only in the rRNA fragments, and the adapter now has a ds restriction cut site in it that can be chopped, making the fragment un-amplifiable by PCR.

[nucacidhunter]

For low input RNAseq without removing rRNA there are two options:

1- For mRNAseq one can use Clontech SMART-Seq Ultra Low Input kit or home brew SMARTseq2: (http://www.nature.com/nprot/journal/....2014.006.html). These use oligo dT for priming 1st strand and are suitable for pg-ng starting total RNA resulting in a library from polyA transcripts.

2- As mentioned by SNPsaurus NuGEN InDA-C technology (10 ng input RNA). This technique is also suitable for library prep from total RNA and prepares stranded libraries from total RNA-rRNA (or any other unwanted transcripts) fraction of transcripts.

[eab]

what if the RNA is degraded, and there's just a tiny bit of it? think SMARTseq2 on a single cell laser captured from an FFPE section.
ever try SMARTer on degraded RNA, as a 3' end sequencing approach?

[nucacidhunter]

what if the RNA is degraded, and there's just a tiny bit of it? think SMARTseq2 on a single cell laser captured from an FFPE section.
ever try SMARTer on degraded RNA, as a 3' end sequencing approach?

SMARTseq2 may not be the best choice for degraded RNA because only 3' end of transcripts will be converted into library.

With current technologies library prep from compromised low input RNA will not give optimum results but depend on study aim one might accept comprise in some aspects. Some options:

1- SMARTer Stranded RNA-Seq Kit can be used but sequencing cost will be high because it does not eliminate rRNA
2- NuGEN technology with lower input
3- SMARTer combined with exome capture (for human samples or model species with established exome capture kit)