I performed a Chip-Seq analysis using a pol II antibody and it yielded some interesting sequences where this protein is accumulated. In order to check if these sequences could be acting as transcription promoters we are amplifying and inserting them upstream a reporter gene (luciferase) in expression vector for our model organism. So far, we have not interesting results suggesting promoter activity, but before rule this out I would like to test alternative options. Can anyone recommend some other experiments for this purpose? It has been a lot of work to give it out so soon!
Hi Carlos,
the presence of Pol2 usually marks promoters and also enhancers, but doesn't tell you much about their activity state - in some cases Pol2 will just sit there and wait for the appropriate signal to initiate transcription. The first thing I'd try is to get independent support from either RNA-seq or CAGE-seq (sequencing of 5'-capped RNAs only) to see whether transcripts are initiated from your Pol2 binding sites. Also, without such data it will be hard to estimate the direction of transcription - did you clone your reporters in both directions or only in one?
Cheers
Hi, Michael, thanks for your answer. I cloned the reporters in both directions, but I am still awainting the results from one of them.
Cheers
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