Input DNA smaller than ChIP DNA

[ash_5760]

Hi all,
I will be doing ChIP-seq for several transcription factors and I am in the ChIP process now. I have done sonication using the covaris S2 along with microTubes w/AFA that hold 130ul. I sonicated 1x10^7 cells in 130ul and did a time course at 10% duty cycle, 200 cycles/burst, intensity=5. I ended up using an 8min sonication with these conditions to shear to ~200-800bp fragments. I did ChIP for 2 different TFs in parallel and when I ran them out on the bioanalyzer, I saw that the input was sheared to 200-300bp but the ChIP peaks were from 2000-7000bp. I am wondering why I am not pulling down anything in the 200-800bp range? I attached the bioA image where the first 8 lanes are the ChIPs and lanes 9 and 10 are the inputs. I am planning to make illumina libraries and sequence samples so my concern is that when I do the size selection step using the TruSeq ChIP kit, I will not have a very complex library (if I have one at all). I would really appreciate any help on this. Thanks!

[Chipper]

The BioAnalyzer does not have the sensitivity to detect unamplified ChIP samples in most cases.

[ash_5760]

So then what is the high molecular weight band?

[Hamid]

Hi Ashely,

As we discussed on the phone, you are:
1. using too many cells for the microTUBE. The microTUBE is designed for only processing nuclei from 3 million cells or less in the 130ul volume. You will need to process 10^7 cells in 1ml volume using our 12x12 tube.
2. you are using the wrong settings. when processing in microTUBEs, we have optimized a setting of 2%duty cycle/3 intensity/200cpb for the initial time course.

Thank you

Hamid

[ash_5760]

Hi Hamid,

If your "optimizations" worked, there wouldn't be any issues...as we discussed. Repetition in a forum is not helpful.

-Ashley