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I am new to ChIP-seq and wanted to get an idea of what protocols are working best for everyone. I have tried the one published by Farnham et al and a variation of it using a Covaris protocol (Chromatin shearing with SDS detergent buffer). In both cases, I am getting less chromatin than what I would have expected from 10^8 U937 cells. I have never gotten back more than a 1 ug following shearing and suspect something is awry. Any thoughts? I suspect perhaps I am losing the bulk of my chromatin at one of the steps or am not getting adequate lysing of cells. Any tips on what has worked for others or tips on might be going wrong would be much appreciated.
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Hi,
1. in what volume are you lysing your cells?
2. What is your fixation time, volume?
3. In what volume are you processing your samples on the Covaris?
4. What is the setting you are using?
5. Are you preparing nuclei prior to chromatin shearing?
6. You should save the cell debris after shearing, and run a portion of it on the gel as a way to check for incomplete lysis/nuclei prep, and shearing.
7. Which type of tubes are you using for the chromatin shearing step on the Covaris?
Would you be interested in receiving the Covaris chromatin shearing buffers from us?
Thank you
Hamid -
Hi Hamid,
1. I am lysing at a concentration of 1 mL lysis buffer/1x10^7 cells
2. My fixation time (cross-linking time) was 10 min. The volume varied but final concentration of formaldehyde was 1%.
3. I had been mostly shearing in 0.5 mL within TC12 tubes
4. The settings are 20% duty cycle, 8 intensity, 200 cycles/burst
5. I had been lysing for 15 min. in 5mM PIPES pH8, 85 mM KCL, 1% IGEPAL, and protease inhibitors. Then I dounce homogenize for 20 strokes and spin at 400 rcf. I reconstitute in shearing/nuclei lysis buffer (50 mM Tris-HCl pH8, 10 mM EDTA, 1% SDS, and protease inhibitors) incubate for 30 min. and then shear for 30 minutes at the settings above.
Today I had luck and got about 700 ug of chromatin from 7x10^7 cells in two different preps. It must have been magic or because I was measuring my DNA concentration via Qubit at every step and with the supernatant and sample. I will see how my shearing went tomorrow. I'm not sure what I did differently this time.
Thank you very much for your response!Comment
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Hi,
In the course of optimizing our protocol, we have found the following:
1. optimization of ixation time is important, and so is the freshness of the HCOH stock solution you are using.
2. Fixation should be carried out in another buffer instead of media.
3. Efficient lysis and nuclei prep is essential in reproducible results.
4. You must carry out a time course for the shearing step i order find the best processing time for your cell line, cell number, and shearing volume.
5. When you run your time course processing on a agarose gel, you should notice a gradual reduction in distribution and size range. If you notice the generation of a 200-300bp band without a gradual reduction, you are over processing the samples.
Let me know if you want to evaluate our validated protocol and buffer formulations. I can send you our new pre-made reagents.
Thank you
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