[ZWB]

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.

http://www.ncbi.nlm.nih.gov/pubmed/23558773

[carmeyeii]

Thanks, kcchan!
That sums it up

[kcchan]

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!

Carmen

Illumina has three main kits on the market for RNA library prep.

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.

[carmeyeii]

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!

Carmen

[frozenlyse]

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.

[jazz]

From my results, Superscript III doesn't need ActD to eliminate spurious antisense strand generation, whereas a lot of other RT enzymes do.

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.

[frozenlyse]

Protist asked "Have you tried including actinomycin D in your second strand synthesis." We use the NEB second strand kit without modification, and it lacks AcD. Do you see a difference in libraries produced with AcD?

When I did the initial library testing we didn't see a noticeable difference and the Parkhomchuk NAR paper I adapted the method from stated "In our hands, the presence of actinomycin D in the reaction did not significantly influence the level of antisense transcription". We have not routinely included actinomycin D in our library preps.

From my results, Superscript III doesn't need ActD to eliminate spurious antisense strand generation, whereas a lot of other RT enzymes do.

[protist]

pbluescript posted a very nice image showing the effect of altering the ratio of AMPure beads/sample in a Primer dimer problem thread - http://seqanswers.com/forums/showthread.php?t=10817. I have reattached the informative image pbluescript added illustrating the effect of altering the ratio of AMPure beads/sample.

[jazz]

While we are discussing the usability of different reagents in preparing libraries, can anyone help me with my issue with Ampure beads. I just ordered them and wanted to test them for size retention using a 100bp ladder. I tried 3 different concentrations- 0.5:1, 1:1 and 1.5:1 for beads to DNA. All three cases, there was no loss of any DNA whatsoever. My understanding is that lower concentration of beads should cause selective retention of large sized DNA (and hence loss of smaller bands). I incubated with the beads for 15 minutes. Is that too much? Can anyone share their experiences/suggestions?

[protist]

Protist asked "Have you tried including actinomycin D in your second strand synthesis." We use the NEB second strand kit without modification, and it lacks AcD. Do you see a difference in libraries produced with AcD?

When I did the initial library testing we didn't see a noticeable difference and the Parkhomchuk NAR paper I adapted the method from stated "In our hands, the presence of actinomycin D in the reaction did not significantly influence the level of antisense transcription". We have not routinely included actinomycin D in our library preps.

[treebeard]

Hey all - couple comments:

1. Protist asked "Have you tried including actinomycin D in your second strand synthesis." We use the NEB second strand kit without modification, and it lacks AcD. Do you see a difference in libraries produced with AcD?

2. Eliminating unincorporated dNTPs. This is the reason we used ammonium rather than sodium acetate for preciptation - it is more effective at removing nucleotides. For complete removal, I think that protist has the best suggestion (e.g., G50 columns).

[perrinwang]

In my experience, quite a bit of dNTP/NTP make it through an AMPure.

Hmm... that is interesting to know... also troublesome for us. I am wondering how much dNTP is retained through Ampure purification, do you have a good guess at percentage?

Here is our protocol: http://dx.plos.org/10.1371/journal.pone.0026426, i think we would have the issue if dNTP is carried over.

[protist]

For the past ~2 yrs all of our RNAseq experiments have been strand specific (dUTP method) - we have consistently used g50 columns for 1st strand clean-uo and rarely see problems with the resultant data.

[ECO]

In my experience, quite a bit of dNTP/NTP make it through an AMPure.

[pmiguel]

We suspect that there must be a small amount of carry-over of dTTP in this method, as only 95% of reads map to the expected strand (and we don't believe that the remaining 5% is due entirely to the presence of antisense RNA).

Any reason not to use AmpPure beads after first strand synthesis? Might be more effective at eliminating the dTTP than an ethanol precipitation.

--
Phillip