SMARTER Pico RNA-seq kit - new ribo-depletion technique

[luc]

The SMARTER Pico RNA-seq kit seems to implement a new ribo-depletion technique - ribodepletion of an almost complete sequencing library. (http://www.clontech.com/US/Products/...10020:22372:US).

Has anybody tried this kit or perhaps has an explanation how it could work?
The ribodepletion happens here after a some initial PCR amplification of the library, thus it should be double-stranded at this point. The they add some reagents (ZapR; likely including a bait library) and incubate at 37C one hour and at 72C for 10 minutes. During this treatment the rRNA-sequence containing library fragments are supposedly cut and thus will not be amplified in the following round of PCR which adds the complete Illumina adapters.

[Simone78]

Originally Posted by luc

The SMARTER Pico RNA-seq kit seems to implement a new ribo-depletion technique - ribodepletion of an almost complete sequencing library. (http://www.clontech.com/US/Products/...10020:22372:US).

Has anybody tried this kit or perhaps has an explanation how it could work?
The ribodepletion happens here after a some initial PCR amplification of the library, thus it should be double-stranded at this point. The they add some reagents (ZapR; likely including a bait library) and incubate at 37C one hour and at 72C for 10 minutes. During this treatment the rRNA-sequence containing library fragments are supposedly cut and thus will not be amplified in the following round of PCR which adds the complete Illumina adapters.

very interesting, thanks for sharing!
from the manual it seems they have a buffer, an enzyme and a set of oligos that are specifically binding the rDNA. The oligos must be RNA oligos (they need to be stored at -80). They might anneal the rDNA to form RNA/DNA hybrids perhaps? In that case the enzyme would be RNAse H. Could it be something similar to this?
PMID: 22900061
In the paper, a set of DNA oligos was hybridized to the rRNA before any amplification, though. This is the same (or very similar) set of oligos that Clontech has in its RiboGone kit.

[nucacidhunter]

Originally Posted by luc

Has anybody tried this kit or perhaps has an explanation how it could work?

This sounds similar to “Insert Dependent Adaptor Cleavage (InDA-C)” that has been adapted to SMART technology. This technology was released in some RNAseq kits early this year. Its performance is not as good as RNAseH or hybridization based methods (according to manufacturer data), but is one the post-cDNA synthesis methods for reducing rRNA reads in libraries with ultra-low inputs which former methods are not applicable.

Edit:
Comparison of protocols indicates that different mechanisms are used in each method. InDA-C method hybridises carefully designed oligos to ssDNA, extends and then uses BspQI (recognition site: GCTCTTCN^NNN) that cleaves adapter making that ssDNA un-amplifiable.

[luc]

Originally Posted by nucacidhunter

This sounds similar to “Insert Dependent Adaptor Cleavage (InDA-C)” that has been adapted to SMART technology. This technology was released in some RNAseq kits early this year. Its performance is not as good as RNAseH or hybridization based methods (according to manufacturer data), but is one the post-cDNA synthesis methods for reducing rRNA reads in libraries with ultra-low inputs which former methods are not applicable.

Hi nucacidhunter,
On the first look it sounds similar, but the InDA-C process requires denaturation of the ds library to get the ribo-sequence oligos to anneal; the Pico RNA-seq kit strangely does not?
(As mentioned below: Perhaps the library is only amplified with a single primer in the first step and thus mostly single-stranded? This might allow for InDA-C style extension and digestion?)

[luc]

Hi Simone,
thanks for your thoughts. I have not found any mention that RNAse H can be somehow motivated to actually digest DNA, which it would need to do in this protocol.
Somehow they induce a sequence-specific destruction of the library molecules (using some kind of bait sequences) without denaturing the library. Perhaps the library is only amplified with a single primer in the first step and thus mostly single-stranded? Otherwise recombinase would perhaps be an option?

Originally Posted by Simone78

very interesting, thanks for sharing!
from the manual it seems they have a buffer, an enzyme and a set of oligos that are specifically binding the rDNA. The oligos must be RNA oligos (they need to be stored at -80). They might anneal the rDNA to form RNA/DNA hybrids perhaps? In that case the enzyme would be RNAse H. Could it be something similar to this?
PMID: 22900061
In the paper, a set of DNA oligos was hybridized to the rRNA before any amplification, though. This is the same (or very similar) set of oligos that Clontech has in its RiboGone kit.

[Simone78]

Originally Posted by luc

Hi Simone,
thanks for your thoughts. I have not found any mention that RNAse H can be somehow motivated to actually digest DNA, which it would need to do in this protocol.
Somehow they induce a sequence-specific destruction of the library molecules (using some kind of bait sequences) without denaturing the library. Perhaps the library is only amplified with a single primer in the first step and thus mostly single-stranded? Otherwise recombinase would perhaps be an option?

You are right! I read the protocol in detail and I was also wondering that there is no denaturation step. Anyway, being interested in single-cell RNA-seq I also realized that this protocol is probably not for me. They say that one needs at least 250 pg of clean RNA. While the protocol can probably be scaled down to single cells, I will never be able to get clean RNA, the genomic DNA will always be present (DNAse treatment on single cell seems not feasible), so the random hexamers will amplify it along with the tot RNA.

[HESmith]

I wonder if the mechanism is triplex DNA-mediated cleavage by poisoned/crippled topoisomerase. The PCR amplification that precedes rRNA removal is only five cycles and includes both forward and reverse primers, so it's not producing ssDNA. Triplex oligos can provide target specificity, and the trapped DNA/topo intermediate blocks amplification in the second PCR. Just guessing, but it's consistent with the protocol.

[luc]

Thanks HESmith!
I will have to read up on this.

Originally Posted by HESmith

I wonder if the mechanism is triplex DNA-mediated cleavage by poisoned/crippled topoisomerase. The PCR amplification that precedes rRNA removal is only five cycles and includes both forward and reverse primers, so it's not producing ssDNA. Triplex oligos can provide target specificity, and the trapped DNA/topo intermediate blocks amplification in the second PCR. Just guessing, but it's consistent with the protocol.

[nucacidhunter]

Originally Posted by HESmith

I wonder if the mechanism is triplex DNA-mediated cleavage by poisoned/crippled topoisomerase. The PCR amplification that precedes rRNA removal is only five cycles and includes both forward and reverse primers, so it's not producing ssDNA. Triplex oligos can provide target specificity, and the trapped DNA/topo intermediate blocks amplification in the second PCR. Just guessing, but it's consistent with the protocol.

Or it could be the method described in this paper: http://www.febsletters.org/article/0...307-9/abstract

[luc]

Thanks Nucacidhunter,
RNAs are scary complex/capable.

[BioKiwi]

There is some anecdotal evidence that libraries prepared with this kit in input range (250pg-10ng) do not show any correlation with 100 ng or higher input libraries prepared with the same input total RNA using TruSeq kit, even though libraries prepared with the kit show good concordance with each other. I wonder if someone has done any comparison and how one can interpret result of a gene expression if it shows different values depending on what kit was used for library prep.