5' end tags

[biochembug]

Hi Friends

We are interested in the 5' ends of the transcripts. I was thinking of setting an RNA adapter at the 5' end (as in directional sequencing), then shearing the RNA with Mg+2 at 94degC for 5 min, ligating another 3' end adapter after T4PNK based 3' end repair, and followed by rest of the methods to get the 5' end of the transcripts.

My question is based on shearing, which is random on any RNA (correct me, if I am wrong!). Upon doing so, will I loose 5' ends of the transcripts?

I think, people who are doing whole RNAseq following Mg+2 based shearing can answer this question based on their read mapping. Are 5' reads low in counts as compared to the 3' end or middle part of the transcript?

Biochembug

[Dario1984]

Why not do CAGE sequencing ?

[biochembug]

Thanks Dario. We already thought of a similar CAGE technique exactly from RIKEN labs, however due to the number of steps we were kind of reluctant. Anyhow, I would like to get an answer for my query. Lets see what others say!