Shearing DNA for Chip-seq

[macgabhain]

Has anyone had trouble shearing formaldehyde crosslinked chromatin to small enough fragments to go into Illumina library prep? Our protocol calls for shearing to 200-300 bp fragments after crosslinking but it seems that we can't breakup the chromatin to anything less the ~800 bp by Bioanalyzer. We have tried traditional sonicator and Covaris. What are we doing wrong?

[Hamid]

Hi,

I wonder which protocol you used to shear chromatin using Covaris? I have attached our two updated protocols. One is for using SDS based buffers, and one is for using non-ionic detergent based buffers. Please prepare your samples according to one of the protocols, and let me know how it works for you.

Thank you

Hamid

[macgabhain]

Thanks, we had tried older protocols from Covaris dated late last year for the SDS buffer. We will try the new non-ionic buffer protocol and post our results as soon we have something.

[Hamid]

Hi,

The new SDS-based buffer protocol works just as well now, so you are welcome to try either protocol. My suggestion would be to use a sample volume of 500ul in the TC12 tubes.
What is the cell type, and cell number you are using in this experiment?

Thank you

Hamid

[macgabhain]

we used the SDS-based buffer protocol with sample volume of 500ul in the TC12 tubes. They were Jurkat cells, and 20 million cells in each tube. We did 5,10,15,20,25 and 30min, with the intensity 5. No difference in shearing size. All stayed around 800-1kb by Bioanalyzer tracing.

[captainentropy]

To me it sounds like you've overcrosslinked your chromatin. What is your crosslink strategy?

Also, I know with the bioruptor the tube choice makes a difference. Some tubes barely let any of the sonic energy through the tube wall. The TPX tubes from diagenode so far are the best. Since the TPX plastic is used in high sound frequency applications so it makes sense that it is the best.

[macgabhain]

1% Formaldehyde for 10 min at room temp, quench with glycine. We are using the tubes recommended for the Covaris. Thanks for your input.

[captainentropy]

hmmm. Interesting. We crosslink (thymocytes) at 1x10^6 cell/mL in 1% formaldehyde for 10 min in a 37 degree air incubator with rotation. Then quench with 0.125M glycine for 10 min on ice.

Your conditions seem mild enough. But I can't help but think they are somehow overcrosslinked. Have your conditions worked in the past and give you a more reasonable 300bp avg frag size?