I need to do ChIP-seq for a transcription factor in human placenta. I have done it dozens of times, changing reagents and experimental conditions, but I have not yet achieved satisfactory fragmentation. I need fragment size ranging from 150-300bp. I have no chip experience and if anyone can help me to get better results, I will be really grateful.
My protocol:
Weight of 30mg of tissue, pulverization using pistil and liquid nitrogen, Add 1ml of PBS1X with 10ul protease inhibitor (100x), and use a syringe and 18G needle for dissociation. I transfer the tissue in PBS to a 15ml tube at room temperature. Add 1% formaldehyde for 10 min at room temperature, inactivate with 0.125 M Glycine on ice for 15 minutes, centrifuge to pellet and freeze overnight. The next day I do cellular and nuclear lysis with protease inhibitor and then sonication in Biorruptor (5 cycles of 30sON/45Off) with separate tubes for diferent pulses (0P, 2P, 5P, 10P, 12P, 15P). After that, I de-crosslink with RNAse A (30min) and Proteinase K (3hr) in a thermal cycler, all in one step. I run on 2% agarose gel. I do not purify or quantify the sonication product. As you can see on the gel (two figures attatched) is pretty commom to have the smear bellow 500bp, even the pulse 2.
P.s.: Can it be the amount of initial tissue? This tissue was collected a nine months ago, snap-frozen in liquid nitrogen and stored at -80.
Phenol:chloroform purification may improve the resolution of the bands. Yet 15P conditions do not seem very bad. The bulk of the smear is below 500 bp. Glycine should be fresh to quench the crosslinking.
I did ChiP experiments around 2005. I established an easy-to-use protocol. If you want try that, please check the protocol described in the reference (Chen KG et al., Cancer Res. 2005; PMID: 16230402). I would be happy to discuss with you if you have any questions after reading my protocol.
Danielle Brotto If you purify the DNA and run the gel, the band will look better. I usually found smear band if the DNA is not pure. If you do not purify the the DNA, then you cannot decide which P is optimum for sonication. Thank you!
Thank you all for the help. I did purified and non purified samples, however, I had no smear or band. I'm going to repeat the experiment.
Aslihan Temel The Glycine I use is from the Magna Chip HiSens Kit from Merck. I think I should have no problem with it.
Kevin G Chen Thank's a lot! I am checking the protocol.
If you are going to purify an aliquot of the IPed sample, try not to lose DNA pellet after EtOH precipitation. In order to obtain a visible pellet, you can also add glycogen in addition to salt and EtOH.
You should try with more than 30 mg. It's very little. How many ng DNA have you loaded on the gel?
I keep fingers crossed I am working on optimization of sonication myself:)
Hi Joanna Bem , sorry for the late reply. I did increase the weight of start material. Right now I am working with 200mg of tissue. With 100 mg worked well, but the reason for having increased even more is that in the end of Immunoprecipitation I dont have enough material. In nanodrop I get around 4ng for RNApolII (positive control), for instance. The fluorometer Qubit don't even measure (out of range). I doubled the antibody amount (1 to 2ug) and nothing change, I don't now what is happening. I am doing qPCR with GAPDH primers, and the Ct values are above 30 for all of them (RNApol, IgG, Input, and my TF).
I am using magnetic beads. Attached is the fragmentation gel of my sample, I think it is ok, but if anybody has something to say, I am open.
Thank you all for the comments, always very useful!