ChIP: Inconsistent reverse crosslinking and DNA purification result - (Sep/03/2014 )
I am a beginner with ChIP. I am in the midst of optimizing my sonication. I think I found the optimal sonication procedures(5 cycles of 30on/30off), but my result is not consistent. When I run reverse-crosslinked and purified DNA on a gel, 50% of time I see a perfect smear from 200bp~1kb but for other 50% time, my smear is usually from 6kb~10kb. I have attached the sample image. For example, the first two samples worked fine but the last one didn't. Even though they went through exact same reverse-cross link and purification step side by side.
I think this inconsistency comes from my de-crosslinking and DNA purification steps and not during the sonication.
My reverse-crosslinking and DNA purifcaiton steps are briefly like this,
Mix 100uL shearted chromatin +100uL EB
65C reverse cross-link with NaCl o/n
Proteinase K mix (Glycoblue+Protease K+TE) treatment for 2 hrs at 42C
1x Phenol/chroloform/iso-amylalacohol extraction, 2x Chloroform extraction
Add 200mM final NaCl and 2.5 volume 100% EtOH. Incubate -80C for 30 min
Spin max for 20min
Wash 2x with 70% EtOH
Air dry 10 min
RNase A treatment for 2 hrs at 37C
Purify using QIAquick PCR purification kit
Any suggestion? Idea?
I have several suspects I could think of:
#1) Is -80C incubation in 100% EtOH for 30 min too long? After 30 min, the solution is usually frozen (I need to warm it up with my hand to turn it into liquid phase before spinning) Is this bad?
#2) When I add RNASE A to the air dried pellet, is it okay to resuspend the pellet by pipetting up and down? Or do you leave the pellet resuspend naturally in 37C block?
#3) When you wash your pellet with 70% EtOH, do you resuspend the pellet? Or do you just tap gently several times
Thank you in advance!
#1) You can switch to -20 C for 30 min if you don't want a frozen tube.
#2) You can add RNase A before Proteinase K digestion so you only need to clean up once with PCI.
What are you using for sonication? Were these samples treated at the same time? It seems more like decreased efficiency (misplaced probe, burned samples, etc.) than a reverse crosslinking issue. The 65 C overnight should have taken care of crosslinking. I'm not certain, but nucleosome content shouldn't shift the distribution by 6 kb... I usually mix my input with buffer containing 1% SDS for overnight crosslinking--just dilute to 2x volume for digestions.
Thank you for your reply.
I am using Diagenode Bioruptor with 4C water bath. Yes, the samples were treated at the same time side by side. I don't think I have a terrible hand, so I don't know where my inconsistency or decreased efficiency is coming from.
Mean while I've tried a shorter protocol from Grant Barish's Lab and it works fine. But I still want the traditional o/n protocol I wrote above to work for me also. I am still figuring out what's the problem...:
Add 3x 100% EtOH
-80C for 15 min
Spin 12,000gx1min 4C
Air dry DNA
Add 100uL Buffer EB and boil for 10 min
Centrifuge 12,000gx1 min 4C
Add 1ul Proteinase K (20mg/ml)
55C for 30 min
Boil for 10 min
Centrifuge 12,000gx1min 4C
Collect top 80ul supernatant to new tube.
Add additional 120uL ddH2O, vortex, centrifuge again and collect the top 120uL supernatant to that same tube.
Purify with PCR kit