Hi Hamed!  I haven't tried ChIP in yeast but as far as mammalian cells are concerned, there could be many reasons responsible for less lysate obtained.

First of all is whether you have started with enough cells. I had this problem initially and to troubleshoot it, I increased the initial number of cells from 2 million to 4-5 million. 

Secondly, as you have pointed out, cross-linking (and reverse cross-linking while chromatin estimation) can contribute to errors while estimating the efficiency of lysate preparation. In case of mammalian cells, 20 minutes of cross-linking using 1% formaldehyde and 5-6 hours of reverse cross-linking at 65 degrees celcius with proteinase K and RNAse is enough.

Thirdly, I am not clear if you quench the cross-linking reagent after the mentioned time (11-16 hours). In case you don't, make sure this is done. Presence of active cross-linking reagent for extended time duration can contribute to incorrect estimation.

And obviously lastly, proper phenol-chlorform purification of chromatin DNA is equally important.

P.S: Check the link from Abcam website

Hope this helps.

Best,

Tapan

http://www.abcam.com/ps/pdf/protocols/x_CHip_protocol.pdf

Hello dear tapan,

Many thanks for your replay and for attaching the file.

I have to do a prolonged double cross-link because the minimum time for second cross-link (which is Formaldehyde) is 10 to 11 hours to get maximum binding of HDACs ( Siavash Kurdistani lab I attached the paper).

I normally use 100 ml of 2X10^7 cell/mL when pull down other proteins like  Rad7 protein and got enough amount of chromatin. in this particular experiment I increased the volume 3 times that is 300 mL still didn't get sufficient amount of chromatin/ DNA. 

Finally, I managed to overcome this problem and I found out that if you increase the volume of lysate  buffer and decrease glass-beads this will lead to increase the yield of chromatin. Before I used 500 micro-liter of lysate and 500 micro-liter of glass beads for each 100 ml cells after pelleting in 2 mL round bottom tubes now I use 750 to 800 micro-liter of lysate buffer with 350 to 400 micro-liter glass-beads.

In summary 

1-I use 200 ml of cells 

2- increased volume of laysate buffer (+ protease inhibitor)

3- decrease galss-beads

thanks once more for your contribution,

Hamed