8 replies to this topic

[TracyDuke]

I am going to do a ChIP against H3K27me3 in a cell type hasn't been studied for this histone modification before. Is there any well recognized common H3K27me3 target. So that I can validate the ChIP assay itself before jumping into the following seqencing step?

[BioBus]

Hi Tracy,


One reply in this forum or other ChIP place did mention that in -2007 nature
papers addressed H3K27me3 as Mark;
Sorry,  for the moment , I cannot remember these papers (authors/ titles);
maybe later.  But  Just for  as neg control withH3K27me3 (i.e. silencing )  ,
for sure , one guy used H3K27me3 on GAPDH promoter.

Bus

[TracyDuke]

Thank you BioBus. If you, by any chance, recall the information about that paper later, would you pls let know? A million thanks!

[BioBus]

TracyDuke, on Jun 29 2009, 12:45 PM, said:

Thank you BioBus. If you, by any chance, recall the information about that paper later, would you pls let know? A million thanks!

I am not sure , but  have a look at ,  Tarjei S. Mikkelsen1,2, ....Bradley E. Bernstein1,4,6* , 2007 nature

http://www.nature.co...ature06008.html

[Clare]

TracyDuke, on Jun 29 2009, 07:27 PM, said:

I am going to do a ChIP against H3K27me3 in a cell type hasn't been studied for this histone modification before. Is there any well recognized common H3K27me3 target. So that I can validate the ChIP assay itself before jumping into the following seqencing step?

We use Gsx2 (a gene involved in the brain) as our positive control. What cells are you using?
Clare

[TracyDuke]

Thank you Clare. We are using B cells.
Say, CD2 is silent in B cells. Can I just use CD2 as a positive control in the validation of H3K27me3-ChIP? If yes, shall I just amplify the core promoter region (spanning the transcriptional start site) of CD2 gene?

[KPDE]

TracyDuke, on Jun 29 2009, 11:27 AM, said:

I am going to do a ChIP against H3K27me3 in a cell type hasn't been studied for this histone modification before. Is there any well recognized common H3K27me3 target. So that I can validate the ChIP assay itself before jumping into the following seqencing step?

Rhodopsin has worked well for us in multiple cell types.

[orlatron]

TracyDuke, on Jun 29 2009, 11:27 AM, said:

I am going to do a ChIP against H3K27me3 in a cell type hasn't been studied for this histone modification before. Is there any well recognized common H3K27me3 target. So that I can validate the ChIP assay itself before jumping into the following seqencing step?

I use SFRP4 - I used it first in a primary cell which hasn't been studied before and it worked really well, it is from:

Schlesinger, Y., et al., Polycomb-mediated methylation on Lys27 of histone H3 pre-marks genes for de novo methylation in cancer. Nature Genetics, 2007. 39(2): p. 232-236 and Sen, G., D. Webster, and D. Barragan, Control of differentiation in a self-renewing mammalian tissue by the histone demethylase JMJD3. Genes and Development, 2008. 22: p. 1865-1870.

Think the supplemental data from Sen has primer sequence.

Good luck

[chipy]

If I plan to use B.actin promoter to see H3K4me3 and H3K27me3 marks, how are the marks suposed to be seen. Should I see no K27 marks??

thanks