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!
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!
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