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Old 03-09-2010, 09:58 PM   #1
Bonn
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Default Shearing Chromatin using Covaris

Hi,
I'm currently trying to use a covaris S2 to shear chormatin to ~200 bp. I'm using 50 million nuclei in 100 Ál buffer in a 6x16mm MicroTube. Using the regular 200 bp protocol (4 min) brings the chromatin reliably down to 200 bases but also strips off the epitopes needed for subsequent IP.
Is there anybody out there with experience on this matter or will I have to optimize the Bioruptor conditions?!
Thanks a lot,
Stefan
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Old 03-10-2010, 08:38 AM   #2
Hamid
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Hi Stefan,

Can you please answer the following questions so I could better help you troubleshoot what you are observing?
1. What is the exact setting you are using?
2. Have you carried out a time course to determine the optimal chromatin shearing condition for your cell line/cell number/tube type?
3. What type of buffer are you using for shearing?
4. How are you processing the sheared samples immediately after shearing for analysis? Can you please provide the detailed steps.
5. Can you please provide a gel image of your time course?

Thank you

hamid
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Old 03-10-2010, 11:05 PM   #3
Bonn
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Hi Hamid,
My initial settings were:

20% DC, 5 In, 200 C/B, 2-6 min

Starting from 4 min I got very nice 200 bp fragments. The problem is that when I IP with this chromatin I can see that enrichment is only 10 fold (as compared to 50-80 fold using a Bioruptor on comparable material) and that enrichment goes down with time of sonication (from 10 fold to ~4 fold for 4 to 6 min). Don't get me wrong, I don't have a problem with the shearing using the covaris, I have a problem loosing epitopes.

I ran some more conditions yesterday where I decreased DC & In and increased time. All of it is down to 200 bp (sometimes even lower) so I'll check IP efficiency today (& tomorrow).

The buffer I'm using is 50 mM HEPES pH 8.0, 10 mM EDTA, 0.5% N-Laurylsarkosyl.

After shearing the material I spin down the residual crud (membrane fragments etc), take an aliquot for checking the DNA quality (size & amount) and I take the rest for IP. IP conditions are standard (PrtA sepharose, RIPA, LiCl etc) and work just fine with the material (D.m. embryonic material).

Did you ever go beyond shearing using a covaris (meaning did you ever IP and QC the results)? Could it be that using 100 kHz in stead of 10-20 kHz just rips of your epitopes (eg histone modifications or TFs).

Thanks a lot for your interest and help,
Cheers, Stefan

P.S.: Gel pics later...
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Old 03-11-2010, 07:31 AM   #4
sole
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Hi Stefan,
I shear my chromatin to a size of 200-300bps using the COVARIS S2.
I started from 40millions cells (lymphoblasts) and I sheared them in 1ml Nuclei Lysis Solution (0,8% SDS) in TC13 glass tubes using this protocol:
Treatment 1:
Duty Cycle 20%
Intensity 6
Cycles per Burst 200
Cycle Time 30 seconds

Treatment 2: (0 stays for minimun allowed)
Duty Cycle 0
Intensity 0
Cycles per Burst 0
Cycle Time 30 seconds

Cycles 60

Treatment 2 permits to retain chromatin crosslinked.
I enclose a gel pic. The last lane on the right (on the left of the last marker) contains purified DNA from the shearing protocol I refer to.

I hope it will be helpful for you.
Here there is also another thread that could help you: http://seqanswers.com/forums/showthread.php?t=3215.

Ah, I ChIPped this chromatin with different antibodies and I obtained a strongless enrichment relative to previous IPs, but still significative. This could also be due to the different sizes of the fragments obtained in the different experiments (in my case the previus were 700-1500bps).

Keep us updated with your experiments
Cheers,
Sole
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Old 03-11-2010, 09:55 AM   #5
Bonn
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Hi Sole,
Thanks a lot for your help. I have a little trouble understanding treatment 2. What do you mean by "Cycles 60" and does treatment 2 follow treatment 1?
In general I got significant IP enrichment using a somewhat similar protocol. The problem is really that you clearly strip off the epitopes the harder you hit your chromatin. The problem that arises is that your "positive" qPCR primers will give you significant enrichment for some known, "perfect" region. Now when you put this sample in the sequencing queue you might loose many "not so perfect" regions, looking in the end at the strongest binding sites.
I'll have some more answers to this around Monday so I'll keep you updated.
Thanks again,
Stefan
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Old 03-11-2010, 12:32 PM   #6
sole
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Hi Stefan,
Treatment 2 follows treatment 1 after every single cycle, it results in a pause between two cycles. Cycles 60 is the total number of cycles that make this shearing a little time-expensive (60 minutes for that size range).

I am new in the field of sequencing and I am preparing to perform my first ChIP sequencing experiment on SOLiD platform.
I see your point for what regards the epitope stripping off and I agree with you that it will end with an understimation of protein binding sites. In my case I precipitate with antibodies for histone modifications, so probably this phenomenon could be negligible in first instance.
I know that using bioruptor you can obtain similar results in terms of shearing but I don't know if it makes any difference in terms of yield of precipitated DNA.

Waiting to hear from you soon,
it's always useful to compare results...
Thanks
Sole
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Old 03-14-2010, 09:20 AM   #7
Hamid
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Hi Stefen,

I apologize for the delay in responding. I have been a bit ill, and not checking the site as much.
To answer you question, we do have customers with chIP-seq and ChIP-Chip data. According to them( Cornell, and USC) the data they obtained was more reproducible. The Cornell data can be found at http://covarisinc.com/chromatin-shearing.html .
I have not yet placed the USC ChIP-ChiP data on the web site since they have kindly agreed to write an application notes for us.
We also do not believe that focused acoustics delivered at 500khz is rips epitopes to a greater extent than sonicators operating at 20khz. Extensive DNA shearing studies using our technology has shown that it is a much gentler process than sonication.
With that being said, I think you might be over processing your samples in the micotubes. Typically such a large cell number is processed in 500ul using the TC12 tubes, and even then a nice range of shearing is seen in about 8-12 minutes of processing. You seem to be noticing 200bp fairly quickly which tells me that you are applying too much energy to your samples using those tubes.
My suggestion would be to reduce the duty cycle to 5%, and also reduce the intensity to 4 and 5, and carry out a time course.
If you take a look at the image shown in thread http://seqanswers.com/forums/showthread.php?t=3215 you can definitely see a shearing range with the time course. The image scan is not very good, and not enough was loaded on the gel, but the smear profile is quite apparent.
I look forward to receiving some gel images via email.

Thank you.

hamid
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Old 03-17-2010, 03:48 PM   #8
TomorrowIsToday
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Hi Bonn,
You are right. It is very important to have both right size and intact epitope binding for chromatin shearing. There is a SOLiD Chip-seq kit launched from Invitorgen last month. You can find a Covaris and Bioruptor condition to shear chromatin into 200-300bp and work well for ChIP-seq application in their online munual.
http://products.invitrogen.com/ivgn/...Search-Product
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Old 11-27-2011, 01:46 PM   #9
Smriti
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Hi Hamid,

I have started some experiments on chromatin shearing recently. As a beginner I wanted to ask, if we have cells prepared of certain cell density in certain vol, can't we use aliquot of that sample to run chromatin shearing? Meaning is it really reqd. to run the entire volume in one batch. Can't we divide sample in parts, shear it and then combine it later?
Also, another thing about reverse crosslinking: Should we always add RNAse and Proteinase K while reverse crosslinking DNA from proteins at 65C? Or just maintaining this temp. is good enough?

Thanks
Smriti
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Old 06-08-2012, 09:50 PM   #10
zhou junfei
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Post covaris S220 fragmented Ribominus RNA

I have fragmented Ribominus RNA(400ng in the 130ul RNase-free water) using S220 followed the method of covaris mRNA and total RNA fragmentation, 175, 10%, 200cycles, 60s~420s, and then checked size distribution on Aglient 2100 small RNA kit. But I didn't recovery the 100~200nt fragments. Please give me some advice to solve this problem.
And , I do this to construct total RNA-seq library using Ribominus RNA for SOLiD 5500.
Another question, does the concentration of RNA is important?
Thank you!
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Old 06-21-2012, 07:46 AM   #11
Hamid
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Hi Zhou,

Sorry for the delay in replying to your post about RNA fragmentation. If you are using the Agilent small RNA chip, you will not see the shearing progress. Please use either the total RNA 6000 nano chip, or the total mRNA 6000 nano chip. Please post the bioanalyzer traces.

Thank you

Hamid
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Old 06-25-2012, 01:37 AM   #12
zhou junfei
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Hi Hamid,
Thank you for your advice. First, I'm so sorry for my lack of knowledge.
I have use the mRNA 6000 Pico chip, but I also feel confusion about these result. However, I found that I just make a mistake about Ribomius RNA, which I thought it must be like mRNA on the chip, but not.
I attacted two traces of two chip, but a question also puzzled me, why the Ribomius RNA peak is more lower than mRNA,which contant mRNA and other species RNA?

Thank you
zhou

Sorry, I didn't attached the traces last time. Now do.


RNA 6000 pico chip
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Last edited by zhou junfei; 07-05-2012 at 04:22 PM.
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Old 08-08-2012, 09:40 AM   #13
wz2145
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Hi Hamid,
I am new to ChIP-Seq. I am optimizing the sonication with Covaris S2, and below is the parameters I used for my last experiment.
Duty Cycle: 5%
Intensity: 4
Cycles per Burst: 200
Tube 6X16 AFA

I did time course 2-12 mins according to the recommendation. But it turns out the sheared chromatin DNA was centered around 1000kp even with 12 mins sonication. I am working on Thp-1 cells, which is supposed to have hard nuclei. Can you please give me some idea what parameter I need to change? Intensity? Time?

Thanks,
Wei
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Old 08-17-2012, 09:08 AM   #14
Hamid
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Hi wz2145,

The settings used is too much for the 6x16mm microTUBEs. The reason why you are not getting the gradual reduction in shearing size range is that you are over-fixing your cells. Since AFA technology is quite different than probe and bath based sonicators, it requires that you follow the fixation, and nuclei preparation according to our protocols and reagents . These reagents and and the protocol have been optimized for AFA based chromatin shearing. The protocol for the low cell kits designed for processing 1-3x10^6 cells in 130ul volume using our microTUBEs can be found at http://covarisinc.com/wp-content/uploads/pn_010145.pdf
Can you please let me know how you are fixing your cells and preparing the nuclei?

Thank you

Hamid
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Old 08-20-2012, 06:22 AM   #15
wz2145
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Thank you for your reply, Hamid.
I use 1.1% Formaldehyde to fix the cells for 15 mins.

For cell lysis and chromatin shearing, I actually used two protocols.

First time, I used Abcam Kit. As I mentioned in the last post, the chromatin did not got completely sheared.

Then, I follow someone else's protocol with two lysis buffer below.

Lysis buffer I, membrane lysis
50 mM Tris PH8.0
140 mM NaCl
1 mM EDTA
10% Glycerol
0.5% NP-40
0.25% Triton X-100

Lysis buffer III, Nuclei lysis
10 mM Tris pH 8.0
1mM EDTA
0.5 mM EGTA

And use the nuclei lysis buffer as chromatin shearing buffer. This time, the shearing worked well, but the chromatin seems degraded after lysis before sonication. There is a very bright band around 1500bp at 0 time point.

Any thought?

Thanks again,

Wei
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Old 08-21-2012, 10:28 AM   #16
vandermj
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I'm having similar problems - my chromatin only gets sheared to about 2kb even at 10 minutes using the settings suggested. I'm using a lot fewer cells - about 100k of FACS sorted tissue. The crosslinking is 1% formaldehyde for 10 min followed by glycine quench and 2 rinses.

I was worried about incomplete cell lysis so I'm very interested to hear how you did it with these two buffers. Can you share more details on that?
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