Illumina directional libraries

[Pilar Z]

Hi,
I am working with an Illumina Genome Analyser GaIIx. I am new at this, and I am studying to produce directional libraries for RNA-Seq. But I have some doubts about what I read in the following article referring to Illumina RNA ligation protocol:
Illumina decaps the RNA with TAP, fragments the RNA with Sodium citrate, then 3' dephosphorylates (with Antartic phosphatase) and then phosphorylates at its 5' end (with T4 polynucleotide kinase) . It don't understand why it does that, because as far as I understand, after decapping, RNA is monophosphorylated on its 5' end so it would be ready for adapter ligation...
Thank you very much for your help,
Pilar

[kcchan]

After fragmentation, the new 5' end of each fragment will need to be phosphorylated with PNK in preparation for adapter ligation. Take a look at this thread which contains a protocol regarding how the process it done.

[Pilar Z]

Hi kcchan,
Thank you for your answer.
Yes I know the 5' end needs to be phosphorylated for the adaptor ligation, but the point of my question is, why on the protocol of Illumina RNA ligation (from the article I mention before), before ligating both 3' and 5' adaptors, it does a 3' dephosphorilation... I ignore the reason why after decapping the mRNA the 3' end is phosphorylated, I've always supposed the 5' end is phosphorylated but not the 3' end, which in turn has an OH...

[Hanabe]

The fragmentation is random, the 'break' can occur before or after the phosphate group that links two nukleotides. That means, that some fragments have the P at the 5'-end, some at the 3', some at both ends and some don't have a phosphate group.

[Pilar Z]

Thank you very much Hanabe, that definitely clarifies my doubt

The fragmentation is random, the 'break' can occur before or after the phosphate group that links two nukleotides. That means, that some fragments have the P at the 5'-end, some at the 3', some at both ends and some don't have a phosphate group.