vic17

Thanks again - really appreciate your advice!

Thanks pcrman - really appreciate your advice. I included IgG only for the qPCR analyses but not for the actual sequencing itself since the chip samples were highly enriched over IgG (in all forms of analyses). And we were told by the core that including the IgG will not make a difference at all. But please let me know if you strongly think otherwise. I might just include an IgG in my sequencing round if it's compelling enough to help reduce background noise (?). I will check the FDR that they've used and ask them to increase the stringency as you suggested. What is the normal FDR cut-off btw? Feel free to advise on any other bioinformatic technique that I could use to yield good signal/noise.

I found the same as well with the Qiagen purification kit. You can actually see the curve (if you are using a nano drop) going from stepwise to smooth and peaks at 260 indicating a more purified sample. I think 60 ug is a little low since you would typically need around 10 ug (lowest I've tried) to more than 100 ug for ChiP. I was having difficulties with a transcription factor - tried twice and it didn't work, and I came across a literature yesterday saying that they've used 150 ug for ChiP with this particular antibody! But then again if your target is abundant , it is worth giving a shot with the amount you already have. good luck and hope it works for you!

No problems, glad to suggest. All the best and happy dissecting!

Hi there, very nice cartoon you've got there but I would use two nice pairs of forceps each time I dissect the zebrafish brain out. The techniques are a little different between fixed vs unfixed whole head/brain, but generally the fixed tissue is more rigid and 'hard' , thus requiring slightly more force when dissecting out the brain. For unfixed head, I would use one pair of forceps for support i.e. to hold the head on an upright position as shown in the cartoon. You can choose to pierce the forceps through the eyes on both sides for better support/grip. WIth the other pair of forceps, gently remove the scales or 'shell' at the top of the head starting from the base/posterior. It's a very hard piece but once it comes off, you should see the dorsal part of the brain almost immediately. Be careful no to pierce through this shell or you would damage the dorsal part of the brain i.e. the midbrain and hindbrain region. This for me is the most challenging part of the process but it becomes a routine after a few practice rounds. The rest of the steps basically involves using a small pair of dissection scissors to cut/detach the optic nerves from the brain - this will need to be done very carefully to avoid damaging the telencephalon. Once done, use the forceps in a closed position to gently 'scoop' the brain from the bottom in a stepwise manner releasing the brain as your forceps move forward. Once you are midway through, you will notice that the brain will start to 'float - out' from the head and a little wiggle with the forceps will release the brain. Just remember to not stretch or compress any part of the brain tissue whilst dissecting.

Hope this helps!