Use antibody accessability as a proxy for PolII accessability.
Compare levels of staining of H3K27me3 in intact vs. mechanically spread nuclei. (First we need to see if H3K27me3 even remains associated in spread nuclei. Try spreading nuclei using the ‘shearing’ lift of a coverglass. Maybe if do this in PBT rather than formamide more proteins will remain attached). Can use H4K16ac as a control for something that should be equally accessible in both conditions.
Why does Pc spread and why does it stop?
- Remove ESC, (PRC2), reduced K27me3 (Esc-like…),
- Repeat in situ + EN stain: no loss of en silencing.
- ChIP en-locus (can use Vivek’s data for K27me3). How about Pc?
- Measure distances between silent loci before and after Pc silencing would have started.
- Remove SCM (non PRC component) by maternal RNAi
- By EN stain, no loss of en silencing. Repeat en in situ (need new DNP probe)
- ChIP en-locus for H3K27me3?
- Measure distance between silent loci before and expected Pc silencing start
- Remove Psc by maternal RNAi
- Stain en RNA and protein. Is expression effected?
- ChIP en-locus