[RamonDT]

Very interesting thread !

Dario, I am also trying to optimized my lysis-sonication protocol and found out that lysis buffer volumen was crucial in my case. I would say you are not lysing properly the cells and therefore you get more DNA released increasing the sonication times. It seems you have more DNA on the wells that could be DNA just released from nuclei by sonication. Just an idea....
Good luck

[dariober]

Very interesting thread !

Glad to see it is useful!

lysis buffer volumen was crucial in my case.

We also think that volume matters and I'm going to try the microtubes.

I would say you are not lysing properly the cells and therefore you get more DNA released increasing the sonication times. It seems you have more DNA on the wells that could be DNA just released from nuclei by sonication.

Yes, I agree. Sonication seems to release DNA from the nuclei. The question is how to improve lysis then.

Thanks for comments!
Dario

[RamonDT]

Hi Dario,

I am not really sure why you are not lysing the cells properly. I am using the Magnify kit and I don't know the composition of the lysis buffer but there is not a nuclear extraction step. May be leaving the samples longer in the lysis buffer? May be trying to disrupt mechanically at the same time? Or may be the detergent in your buffer is not good or is not in the right concentration....
I don't have any clear suggestion by now.

Cheers

[dariober]

SDS buffer for shearing

For those who are still following this thread here's another installment to our chromatin shearing story...

It appears that changing shearing buffer was determinant in obtaining a good shearing. This time the shearing buffer (aka nuclei lysis buffer) was 1% SDS, 10mM EDTA, 50mM Tris pH 8.0. The buffer we previously used did not contain SDS (we were following the Covaris protocol with non-ionic detergent).

As shown in the attached pdf, the shearing now looks good with 10 min sonication. I should say we cranked up the sonicator a bit more compared to previous experiments (20% duty cycle vs 10%, intensity 8 vs 5). However, I still think it was the SDS that made the difference.

Any and all comments, much appreciated!

Dario

[sem]

Any sucess?

Just found this thread. We are considering switching from the Bioruptor to Covaris for chromatin shearing prior to ChIP seq. Have you had luck with IPing from you sheared material and were you able to successfully amplify it for Illumina sequencing?
Thanks in advance for the update!

[dariober]

Just found this thread. We are considering switching from the Bioruptor to Covaris for chromatin shearing prior to ChIP seq. Have you had luck with IPing from you sheared material and were you able to successfully amplify it for Illumina sequencing?
Thanks in advance for the update!

Hi sem,

Yes, eventually we got good shearing and good reads from the sequencer. We are still analyzing the data, but we are quite confident that technically it worked. (However, I have no experience with Bioruptor so I can't comment on how it compares to Covaris).

Good luck!
Dario

[Hamid]

Hi Dario,

Sorry I lost track of the thread. I am glad that your ChiP-seq is working. What was your sample volume for each shearing? If possible can you post your coverage data from your sequencing to show the ChIP efficiency using your Covaris instrument.

We find that the our SDS buffer does work better with most cell lines, but quite a few users would like to avoid such high SDS concentration of 1%. We have managed to reduce our SDS concentration dramatically and still obtain very good shearing results.
I will post some internal BioAnalyzer, western, and qPCR data in the next week or so.

Hamid

[RNAseqer]

anyone find a solution to replace those $5 tubes by Covaris ?

[Hamid]

Dear RNAseqer,

Sample prep does matter to the overall NGS results. The engineered Covaris microTubes to which you are referring are an integral part of the Covaris AFA technology for non-contact, isothermal, highly reproducible DNA fragmentation. If you were to consider the cost of your precious sample, reagents and time for the library preparation and sequencing run, the cost of the microTube is a miniscule percentage.

Thank you

Hamid

[captainentropy]

I've never used the Covaris but I think it's pretty much impossible to mechanically shear your DNA too small. When it gets down to say 150bp you just can't get it smaller no matter how high you crank the thing and how long you sonicate for. That's my experience with tip sonicators.

I have the same view as ETHANol about the shearing limit. Here's a response to an email I sent Covaris regarding this:

You are absolutely right about the size limit of DNA fragments that can be achieved with AFA technology. We believe that the limit is 80 bp (fairly narrow peak with apex around 80 bp). To get such a short fragments you need to shear sample for around one hr. The size limit have probably something to do with the mechanism of DNA fragmentation. The acoustic energy involved in shearing DNA to a small fragments creates some inertial cavitation. In this case bubbles collapse, rebound and emit a pressure pulse or microjet that fragments DNA. The number of bubbles collapsing and their energy could cause the simultaneous fragmentation of the same DNA molecule in two different locations. However, the rigidity of DNA strand, which is closely related to the ability to fragment it, is an exponential function of the inverse of DNA strand length - hence the natural size limit.
I haven't done any experiments with crosslinked chromatin to determine size limit, since most people are interested in fragments around 200 bp, but I suspect that it exists and is probably around 100 bp.

If you have any other questions, please do not hesitate to contact me

Regards,

[Xxxx]


[Xxxx Xxxxxxxxxxxx] Ph.D.
Covaris, Inc.

(I removed the person's name just in case they wouldn't want it reprinted)

[captainentropy]

However, I still think it was the SDS that made the difference.

Any and all comments, much appreciated!

Dario

I agree. I've tested shearing with and without SDS and it was a night and day difference. SDS is a must. We sonicate in 1% SDS. I recall testing 0.1% and it still sonicated ok, but it could be dependent on other factors too to drop down that low.

[yaanlpc]

any one can make more comments?

[theduke]

Also I'd consider using a MNase-based fragmentation protocol. I personally thinks it works better (more even across genomic elements and less damaging to epitopes) then mechanical techniques but it probably depends on your starting material.

Couldn't agree more with this statement. I stopped using sonication a long time ago. And, yes, I work with TFs. The results compared to sonication are worlds apart in terms of quality and reproducibility.

[captainentropy]

Couldn't agree more with this statement. I stopped using sonication a long time ago. And, yes, I work with TFs. The results compared to sonication are worlds apart in terms of quality and reproducibility.

How many nucleosome lengths do you digest down to and still retain your quality and reproducibility?