Chip-seq: Comment on shearing

[dariober]

Hello,

I'm new to immunoprecipitation and DNA shearing. I would really appreciate any comment on the shearing I obtained on two samples I'm preparing (please see attached pdf for gel picture and other details). The downstream application will be Chip-seq sequencing by Illumina sequencer.

Some questions I have:

- Is the size of the smear acceptable for Chipseq?

- Having loaded up to 2 ug, the smear is quite weak. I think a lot of DNA has been shattered in small pieces that ran out of the gel. Is this worrying?

- On the Nanodrop, the two samples have nearly identical concentration (c.ca 360 ng/ul) and have been processed together. Still the smears look quite different on gel. Is this worrying?

Many thanks!

Dario

[pmiguel]

Many substances are optically active at 260 nm. Also UV spectrophotometry is essentially blind to the difference between double-stranded DNA and all other DNA/RNA nucleotides and oligo/polynucleotides.

For this reason, a UV spec reading, such as that given you by your nanodrop, may not be measuring what you want it to measure. In the case of a genomic DNA prep, for instance, it rarely is.

You might want to run an aliquot of your sample prior to shearing on an agarose gel to determine the concentration of what you care about.

--
Phillip

[dariober]

Thanks Phillip for taking care to reply.

Many substances are optically active at 260 nm. Also UV spectrophotometry is essentially blind to the difference between double-stranded DNA and all other DNA/RNA nucleotides and oligo/polynucleotides.

For this reason, a UV spec reading, such as that given you by your nanodrop, may not be measuring what you want it to measure. In the case of a genomic DNA prep, for instance, it rarely is.

Yes, I agree UV spec can be misleading but... 1) The starting material was a cell culture of macrophages, so not many problematic compounds that should have been co-extracted (... I think). 2) DNA was extracted by phenol/chloroform so proteins and other compounds should have been removed (by the way, 260/230 and 260/280 are nicely around >=2).

You might want to run an aliquot of your sample prior to shearing on an agarose gel to determine the concentration of what you care about.

That's right. I thought about doing this when it was too late!

Any comment about the sizing of the smear?

Dario

[pmiguel]

Thanks Phillip for taking care to reply.

Yes, I agree UV spec can be misleading but... 1) The starting material was a cell culture of macrophages, so not many problematic compounds that should have been co-extracted (... I think). 2) DNA was extracted by phenol/chloroform so proteins and other compounds should have been removed (by the way, 260/230 and 260/280 are nicely around >=2).

Alas this is common, even default, presumption. But it is tragically wrong! The contaminant most commonly mistaken for DNA in a spec reading is, of course, RNA.

There is a >95% chance that your response to the above will be: "Not a problem, I used RNAse." Again, tragically wrong! RNAse does not much impact the 260 nm absorbance of RNA. It just breaks the bonds between nucleotides, it does nothing to the bases themselves that absorb around 260 nm. Sometimes a post RNAse precipitation is used to remove the degraded RNA bases. Again, sadly this will rarely be sufficient. There is just too high a ratio of RNA to genomic DNA in cells for a single precipitation to remove enough of it to prevent it from biasing your results.

Then, even if you did get rid of all the RNA, even the tiniest amount of residual phenol can throw you off. Don't believe me? Feast your eyes on this:



This is 0.1% phenol in water. No DNA or RNA at all! 260/280 and 260/230 both >2!

That's right. I thought about doing this when it was too late!

Any comment about the sizing of the smear?

Dario

Sorry, I have never done chromatin immunoprecipitation.

--
Phillip

[ETHANol]

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'd say the fragmentation looks good enough (ideally you might want to get them a little smaller) but the two sizes look different. It will make comparative studies easier if you have the exact same fragmentation for the two samples.

It also looks like your sample may have some salt in it causing the middle samples to run slower. You should run a 2% gel since you are interested in small fragments. I'm not sure.

Quantitate your DNA with QuantIT (Invitrogen). Still not perfect but better then the NanoDrop. And for your starting material, nothing beats electrophoresis.

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.

[dariober]

Thanks guys! Curious to see that the good old (and cheap) agarose gel beats fancier methods!

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.

Can you recommend me a protocol for using MNase for Chip-seq?

Dario

[ETHANol]

http://www.epigenome-noe.net/WWW/researchtools/protocol.php?protid=10

I use MNase from NEB - 1ul for 10 min (15 cm TC plate) but that depends on your starting material.

[Hamid]

Hi Dariober,

I have a few questions which can help us troubleshoot what you are seeing:
1. Are you shearing post IP?
2. What is the size range that you are interested in?
3. In what buffer are you shearing?
the settings you are using, generates 300bp fragments which is often used in the library preparation for the Illumina platform.
4. In what volume and tube type are you fragmenting your DNA?

Thank you

Hamid

[dariober]

Hi Hamid,

Many thanks for offering help! Really appreciated.

1. Are you shearing post IP?

No. We do the shearing on the cross-linked DNA and we precipitate after pre-clearing.

2. What is the size range that you are interested in?

We are going for Illumina/Solexa sequencing. So ideal, as far as I know, is 200-300bp (up to 500bp should be still ok )

3. In what buffer are you shearing?

We shear in buffer LB3 (10mM Tris-HCl, pH 8.0, 200mM NaCl, 1mM EDTA, pH 8.0, 0.5mM EGTA, pH 8.0, 0.1%Na-Deoxycholate, 0.5% N-lauroylsarcosine) complemented with protease inhibitor

the settings you are using, generates 300bp fragments which is often used in the library preparation for the Illumina platform.

Does it surprise you that we get such shearing (see attached in my first post) with only 120 sec on the Covaris?

4. In what volume and tube type are you fragmenting your DNA?

Volume: 2ml. Tubes are Chromacol 5-SV (Chromacol 5mL Screw Top Round Bottom Vial - Clear)

If relevant, the starting amount of cells in each sample is 25-30 million

Thanks again!
Dario

[Hamid]

Hi Dario,

It actually is very surprising that you are getting shearing of cross linked material to that size range in such a short time. Typically with that number of cells, using our protocol, we have to shear using a setting of 20%dc/5i/200cpb/ for 8-15 minutes depending on the cell line.
1. I am not familiar with the tubes you are using at all. Is it a tube you purchased from Covaris or a distributor?
2. You seem to be following our protocol. Are you following it from the beginning, or just at the shearing step?
3. Are yo isolating the nuclei per our protocol?
4. For how long do you cross link?
5. Did you at any point, carry out a time course processing as we have suggested in our protocol?

Thank you

Hamid

[dariober]

Hey Hamid,

It's very kind of you to help me with this!

The protocol I'm following until the shearing step is from Covaris (http://www.covarisinc.com/pdf/pn_400066.pdf).

1. I am not familiar with the tubes you are using at all. Is it a tube you purchased from Covaris or a distributor?

They come from Kbiosciences which should be the UK distributor for Covaris consumables. These tubes are manufactured by Chromacol (https://www.esslab.com/store/5-sv.html) and should be the same of the TC12 on the Covaris protocol.

2. You seem to be following our protocol. Are you following it from the beginning, or just at the shearing step?
3. Are yo isolating the nuclei per our protocol?

Yes, as I said above I'm following the Covaris (http://www.covarisinc.com/pdf/pn_400066.pdf) until the shearing step.
Adherent cells have been crosslinked directly on the plates.

4. For how long do you cross link?

10 min (maybe a bit longer), RT, on rocker.

5. Did you at any point, carry out a time course processing as we have suggested in our protocol?

I haven't done it myself but some collaborators I'm working with did. The bottom line seems to be that even short sonication times give very small fragments.

All the best
Dario

[Hamid]

Hi Dario,

I think I have an idea of why you are getting shearing to such a small size range so quickly from chromatin that should be cross linked. I Here are my suggestions:
1. Please check concentrations of your reagents again to make sure they were made correctly to the concentration we suggest
2. Check the formaldehyde stock solution you used to make the 37% working solution. If it was not fresh, it is quite possible that you did not have the 1% final concentration during your cross linking for 10 minutes. As a result, your cells were not cross linked, therefore it sheared very quickly.
3. What is the cell line that you are using, and at what volume are you processing your samples in the TC12 tubes?
4. Please repeat your experiments with fresh reagents, and please remember to dilute your HCOH to 37% with fresh formaldehyde just prior to use.

Thank you.

Hamid

[dariober]

Hi Hamid,

1. Please check concentrations of your reagents again to make sure they were made correctly to the concentration we suggest
2. Check the formaldehyde stock solution you used to make the 37% working solution. If it was not fresh, it is quite possible that you did not have the 1% final concentration during your cross linking for 10 minutes. As a result, your cells were not cross linked, therefore it sheared very quickly.

I will repeat the experiment making sure the concentrations are correct and the solutions fresh (I would say they were, but you never know...!).

3. What is the cell line that you are using, and at what volume are you processing your samples in the TC12 tubes?

Alveolar macrophages. The volume is 2ml.

4. Please repeat your experiments with fresh reagents, and please remember to dilute your HCOH to 37% with fresh formaldehyde just prior to use.

HCOH was diluted fresh from 37% stock (which was recently bought).
What about the other buffers? How long can they be stored in the fridge?

Many thanks, as usual!

Dario

[Hamid]

Hi Dario,

The other reagents can be kept in the fridge for at least 3 months. You will have to warm up some the LB1, and LB3 prior to use to make sure all the components are dissolved. you then then place them on ice for use. Also please remember to add the protease inhibitors just prior to use.
For the TC12 tubes, I would not suggest using a volume greater than 1.2ml. Typically i suggest using a volume between 500ul-1.2ml for chromatin shearing in the TC12 tubes.

Thank you

Hamid

[dariober]

Shearing time course

Hi Hamid and All,

Hope you are still following this thread...!

As you suggested, we performed a time course on the shearing step. Same settings as before (DC=10, Intensity= 5 Cycles/burst= 200), but shearing for 3, 5, 10, 15 minutes, in duplicates.

As before, we followed the covaris protocol http://www.covarisinc.com/pdf/pn_400066.pdf on adherent macrophages (c.ca 50 million/sample). Please see the attached pdf with the results of the shearing.

We are a bit puzzled by what we found, namely:
1. Increasing time doesn't change the sizing of the smearing which remains between 200-300 bp (which is good size!), with a blob around 100.

2. However, increasing time increases the fragments recovered around 12kb.

3. Indeed, the *total* amount of DNA recovered after phenol/chloroform extraction is correlated with the shearing time (p= 0.0129). Does it mean that most of the chromatin remains un-sheared at short times and is not recovered at all?

Now...
Is it worth increasing the shearing time even more? Is it recommendable to play with the other Covaris settings (duty cycle, intensity)?

(Maybe for another thread...) since we are aiming for ChIP-seq, after having sheared and immuno-precipitated the chromatin would it be an option to elute from the gel the band around 2-300 bp? So to avoid the low and high molecular weight fragments?

Hope to hearing from you!

Dario

Hi Dario,

The other reagents can be kept in the fridge for at least 3 months. You will have to warm up some the LB1, and LB3 prior to use to make sure all the components are dissolved. you then then place them on ice for use. Also please remember to add the protease inhibitors just prior to use.
For the TC12 tubes, I would not suggest using a volume greater than 1.2ml. Typically i suggest using a volume between 500ul-1.2ml for chromatin shearing in the TC12 tubes.

Thank you

Hamid