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Originally posted by frozenlyse
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What are your thoughts about Superscript II? I have seen a few protocols which use Superscript II for first strand synthesis and I think there might be some advantage to it while there is already an improved version of the enzyme available. -
Well, the original paper about ActD ablating spurious antisense signal - http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2095812/ - used Superscript II, so I would consider using ActD or III for directional cDNA synthesis.Originally posted by jazz View PostComment
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I am confused -
Illumina's TruSeq IS actually the dUTP method, while the Standard Illumina TotalRNA is non-stranded, by ligating the adapters after regular cDNA synthesis, right?
What exactly is the Illumina directional protocol?
Thanks a lot!
CarmenComment
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Illumina has three main kits on the market for RNA library prep.Originally posted by carmeyeii View Post
The TruSeq Stranded mRNA and TruSeq Stranded TotalRNA generate directional libraries using the dUTP method.
Meanwhile, the TruSeq RNA v2 kit does NOT generate directional libraries.
There is also another Illumina protocol called Directional mRNA. This version uses a ligation method to determine directionality. This protocol requires both the TruSeq RNA kit and the Small RNA kit or homebrew reagents. Illumina has never released an official kit for this method.Comment
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Thanks, kcchan!
That sums it up
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This is a recently published technique that we developed to make directional RNA-Seq libraries. It's similar to one of the techniques in the Levin et al paper, but we made some significant improvements.
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Hi all,
I started to use Illumina TruSeq Stranded mRNA kit: http://support.illumina.com/sequenci..._prep_kit.ilmn
and I noticed a couple of thing that puzzled me a lot.
First, the protocol for first strand synthesis suggested in the manual is 25C - 10 minutes, 42 C - 15 minutes and 70C 15 minutes. Only 15 minutes, is this really sufficient? Second, there is no purification after first strand synthesis, the second strand mix is added immediately after RT. I always thought that for strand-specific protocol it is critically important to completely eliminate dTTP before second strand synthesis. And last, there is no stage of UDG treatment at all! After adapter ligation and purification the library goes directly to PCR enrichment.
Does anyone have an idea how this could work?Comment
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This is the problem with companies in general. They sell kits with 'proprietary' components and won't disclose any other information.
15' does sound insufficient but maybe they are using a modified version of RT enzyme. However, given that it requires 42C temp, my bet is on Superscript II, which is an older generation. It's up to you if you want to modify the program, since regular RT reactions are 50 min-1hr.
You don't need to purify the Ist reaction products before 2nd strand synthesis. Just modifying the buffer conditions is enough. Check the Parkhomchuck 2009 NAR paper where they described their version of directional RNA-seq. No UDG step is required in Illumina protocol since they are ligating adapters first. This is the major difference between their protocol and UDG based strand detection. If I were you, I would follow the Illumina protocol as is, since it is standardized, and hey, you already have spent a ton of money getting the kist, might as well take advantage of that. Otherwise, have a look at the Parkhomchuck paper, which is what I follow for strand-specific library preps.Comment
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The TruSeq RNA kits don't come with the RT enzyme. You have to supply your own SuperScript II.Comment
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Hi jazz,
indeed, the enzyme used in this kit is user-supplied SuperScript II, as kcchan already mentioned. So 15 minutes looks like a typo.
As for the method, it seems that Illumina uses the method similar to Parkhomchuk's one: "Strand specificity is achieved by replacing dTTP with dUTP in the Second Strand Marking Mix (SMM)": http://support.illumina.com/sequenci...questions.ilmn so the absence of purification after first strand synthesis is still surprising.Comment
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Not sure what that means so....Originally posted by jazz View Post
From the Illumina website.
/truseq_stranded_mrna_lt_sample_prep_kit/questions.ilmn
How is strandedness maintained after DNA amplification?
Strandedness is maintained via the directionality of the adapters. The p7 adapter will be on the 3' end of the cDNA strand. As a consequence, the cDNA strand is sequenced. Second strand synthesis is performed using dUTP in place of dUTP and a high fidelity polymerase that cannot process through dUTP template is used for PCR enrichment. As a result, only the first strand product is amplified and the strand information is retained based on the p5 and p7 adapter orientation.--------------
EthanComment
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We have seen a lower yield with these stranded mRNA kits. More than would be expected from just the loss of the 2nd strand during amplification.
We nearly always fragment for only 4 minutes (instead of 8 minutes) to allow isolation of longer cDNA and produce larger insert libraries. This is because a large percentage of our samples are de novo transcriptome.
Anyway, I had not previously noticed the decrease in the length of the reverse transcription step. We may have been relying on this longer extension to produce our longer cDNAs.
Does anyone think that bumping the RT step back to 50 minutes would cause a loss of strandedness of the resulting libraries? I mean, the SuperScript II will gladly do 2nd strand as well as 1st under normal conditions. The presence of Actinomycin D would inhibit that DNA-templated DNA polymerase activity, but the fact that the step was shortened to 15 minutes worries me. Like maybe a longer incubation allows the SuperScript to circumvent the ActD inhibition?
Thoughts anyone?
Or better, has anyone gone back to 50 minute incubations with SuperScriptII and then observed whether some of the strandedness of the resulting library is lost?
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PhillipComment
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Increasing the time to 50 minutes should not cause a loss of strandedness. Are you doing any additional cleanups with the Directional protocol. If UDG is being added to the polymerase mix, this would result in a lower yield. It would be interesting to perform a typical RNA-Seq library, a stranded library, with the polymerase from the typical RNA-Seq library, then compare yields.Comment
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We don't do additional cleanup, but since dUTP is not added until 2nd strand synthesis, no additional cleanup is actually needed.Originally posted by Genohub View Post
The real problem to maintaining strandedness is just making sure the reverse transcriptase does no 2nd strand synthesis. As long as that does not happen, the libraries should be stranded.
BTW, we use this method now and it appears to work fine -- with the 15' RT incubation. No one seems to think that doubling that incubation time will result in RT doing DNA-templated polymerization.
Spec on the kit is 15 cycles of amplification. We used to typically use 8 cycles. But with the stranded kits we are going to 12-15 cycles. 15 cycles tends to take us about 10 nM, though.
But we scale back the Ampure cuts to mostly remove molecules with inserts smaller than 200 bp. So if the RT is not getting out that far in 15 minutes, that could be eating into our yields.
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PhillipComment
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The original dUTP protocol required massive amounts of starting material because the ActD. If you removed ActD from the protocol you still got pretty good strandedness but required a lot less starting material. So my guess was that the Illumina stranded kit would give a lot lower yield. I'll find out today as I'll be using it for the first time. They do say that you can start out with 4 ug of RNA vs. 1 ug from the unstranded kit so that is some testament to lower efficiency. But I was thinking the yield was going to be a lot lower then that 4-fold difference in requested starting material suggested. From you experience that seems like the case.
I'm not sure why Illumina is recommending the same 15 cycles of PCR for both kits. In my experience with the unstranded kit that was way too many. I guess from your experience for that stranded kit that is more in the correct range.
Phillip sorry to hijack your thread a little, but how are you quantitating your libraries for pooling?--------------
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