Exploring nuclear topology by tracking Pol2
Transcription is central to all life, but many things about it are unclear.
In a nutshell, current methods to research transcription are either addressing factor dynamics in small sub-volumes of the nucleus via imaging, or produce genome-wide snapshots of arrangements of factors in bulk via next generation sequencing (NGS).
In general, there is a disconnect between these approaches. In this project, we want to bridge this gap with a novel assay design that tracks a small number of RNA polymerase II molecules through an in vivo labelling approach.
Our overall aim is to develop this technique and generate data with it for a mammalian cell line followed by analysis using bioinformatics approaches. The main steps will include (i) cloning and (ii) inserting the relevant constructs into HEK293 cells, (iii) verifying the principles of the technique using western blotting and immunofluorescence imaging, (iv) preparing next gen sequencing based datasets such as ChIP-seq and NET-seq, and (v) data processing and analysis