Sequence for directional mRNA seq adaptors?

[gaster]

I am looking for the sequences of the sRNA3', sRNA 5', and SRA Rt primer mentioned in the Illumina Directional mRNA seq adaptor. These sequences are listed in the main "sequence" thread but I noticed the the sRNA 3' listed there lacks the 5' adenylation crucial for ligation using the truncated RNA pol.

Also, does anyone know if these oligos have to be RNA (or have to containe Uracil)? Seems like it is safer and cheaper to make them DNA.

Thanks!

[link1]

the sequences of the adapters and primers for directional mRNA seq are the same as you would use for small RNA library prep. I know the adenylated adapter is DNA.

[gaster]

Thanks for your responses, Link1.

Does the 5' adaptors have to be RNA? It seem cheaper and more stable to use DNA.

[link1]

yes, the 5' adapter should be RNA, otherwise it won't work.

[raafekie]

One step I'm a little hazy on:

In the Illumina directional protocol, when fragmented mRNA is being end repaired, why does the protocol call for using Antarctic Phosphatase and then immediately afterwards PNK. What is achieved by using a 5' phosphatase prior to a 5' kinase?

Thanks!
Joe

[ECO]

AAP removes 3' phosphates also.

[raafekie]

Is the AAP 3' phosphatase activity any different from the 3' phosphatase activity of PNK in the following step?

[ifigeneiastavrou]

Hello,

This is probably a stupid question but i wanna sort it out... I am using the DSN Normalisation protocol by Illumina and at some point it says something about mRNA sequencing and directional mRNA sequencing. Can anyone explain to me the difference between those two?

Thanx