Ion Torrent RNA library prep

[miRman]

Can anybody here explain the molecular biology behind Ion Torrent RNA library prep? The protocol is particularly vague about adapter hybridization and ligation which is what I would like to understand.

According to the protocol, first the adapters are annealed and then ligated. Does this mean that the adapters are partly double stranded or a hairpin?

[miRman]

I found out some details of the prep from Ion Torrent directly. It seems that there are two distinct oligos per fragment end.

See http://www.labome.com/method/RNA-seq...equencing.html for a flow diagram describing the library prep and more details.

[mbzmg1]

Correct -the adapters that are hybridized and ligated to your RNA fragments have degenerate overhangs that allow for strand-specific ligation, and therefore sequencing. As far as I can gather this is essentially the same strategy as older Ambion RNAseq kits (eg http://www.lifetechnologies.com/us/e...equencing.html).

See also the attached slide.

Hope this helps.