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Old 11-30-2011, 04:30 AM   #41
kalyankpy
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Default Share the protocol

Quote:
Originally Posted by ETHANol View Post
After searching the web ways to barcode ChIP-seq libraries, it seems like the best option is to add a barcode at the end of the adaptors (Lefrancois et al 2009) but this requires you to make libraries in multiples of four and the invariant T I'm told may cause issues during sequencing. Illumina still has not released TruSeq for ChIP-seq so I put together a protocol which seems like it should work and uses the reagents we have from the previous ChIP-seq protocol. Illumina has released the adapter sequences but I had to take my best guess at the sequence of the PCR primers.

After spending a beautiful summer weekend in the lab dialing this protocol in, I get good amplification and a nice smear in the correct size range with only 11 cycles of PCR (total cycles from first and second PCR steps) and no self-ligated adaptors. So in theory it looks good.

Has anyone tried ChIP-seq with TruSeq primers? What's your opinion of the attached protocol? Do you think it will work or am I about to waste a thousand dollars on sequencing junk?

Any comments or improvements are appreciated.

A slightly updated version of the protocol is here
http://ethanomics.files.wordpress.co...hip_truseq.pdf

Attached file removed check the updated version on my blog.
Dear Ethanol,

We also tried a chip-seq with trueseq kit without much success. Can u share the protocol with us!

Kalyan
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Old 11-30-2011, 04:47 AM   #42
ETHANol
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Looks like I updated the protocol but not the link.
For now it is here:
http://ethanomics.files.wordpress.co...ip_truseq3.pdf

But if I update it, you can still find it on my blog at:
http://ethanomics.wordpress.com/

It is working great. Finishing it off with the Ampure XP purification gets rid of any extra PCR oligos which if exist in excess inhibit binding of the library to the flow cell.
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Old 11-30-2011, 05:00 AM   #43
kalyankpy
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Default ChIP-Seq protocol

Hi Ethanol,

Thanks for sharing the protocol. I noticed that you are doing end repair and A-tailing in two steps. We have a shortened protocol that combines end repair and A-tailing for this part of the protocol. You may consider it as it worked perfectly for us (Bseq project)

Sonicated DNA = 16 ul
NEB buff #2 = 2 ul
Klenow fragment=1 ul
dNTP mix = 1 ul (10mM dATP; 1mMdGTP, 1mM dCTP, 1mMdTTP)

Mix and incubate at 30 deg for 20 min (end repair) and 37 deg for 20 min (A-tailing).

I also noticed that Myers lab does it at 37 deg for 30 min in one go! It saves time and resources when there are many libraries to prepare!


Quote:
Originally Posted by ETHANol View Post
Looks like I updated the protocol but not the link.
For now it is here:
http://ethanomics.files.wordpress.co...ip_truseq3.pdf

But if I update it, you can still find it on my blog at:
http://ethanomics.wordpress.com/

It is working great. Finishing it off with the Ampure XP purification gets rid of any extra PCR oligos which if exist in excess inhibit binding of the library to the flow cell.
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Old 11-30-2011, 05:06 AM   #44
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Out of curiosity, anyone know how often klenow would add a base other than "A" as a 3' tail to blunt DNA?

I know you can force Taq to add a "T" by having only dTTP available during the tailing reaction. So that makes me think that polymerases with this capability might (rarely) add bases other than A to a blunt 3' end.

--
Phillip
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Old 11-30-2011, 05:19 AM   #45
kalyankpy
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Default Which nucleotide to Add

Hi Phillip,

I dont have a definite answer for you. All I can say is our library worked perfectly fine with just Klenow! It is interesting for me to know if someone knows the answer for your question.

Kalyan

Quote:
Originally Posted by pmiguel View Post
Out of curiosity, anyone know how often klenow would add a base other than "A" as a 3' tail to blunt DNA?

I know you can force Taq to add a "T" by having only dTTP available during the tailing reaction. So that makes me think that polymerases with this capability might (rarely) add bases other than A to a blunt 3' end.

--
Phillip
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Old 11-30-2011, 06:15 AM   #46
jlove
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I'm using the TruSeq kit with ChIP-Seq routinely with good success, if anyone wants to give it a go using the kit instead of homemade components. I just use a 1:10 dilution of adapter for ~ 100 ng of sonicated WCE (or 1:50 for IPs) and do the PCR *prior to* gel size selection. I have a Pippin Prep for size selection.
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Old 11-30-2011, 06:35 AM   #47
ETHANol
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Kalyan,
That is an interesting trick to shorten the protocol. I worry a little bit about the efficiency as Klenow is not that great at blunting ends especially at elevated temperatures. For making plasmids it is always suggested that the incubation be done at room temperature. It clearly works but I wonder what the efficiency is compared to a two step protocol.

Illumina A-tails and then proceeds to ligation without a clean up step. I'd like to give that a try but the protocol is working well so I haven't much of a drive to try it or figure out the conditions. Does has anyone have this working? Perhaps this decreases adapter dimer formation as well.
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Old 12-10-2011, 04:56 AM   #48
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Hi Ethanol,

thanks for sharing your experiences and your protocol. It seems very promising. I am wondering if you have repeated any hiseq runs till now and got more than 50 mio reads? Also, how many clusters you got in the first run, and how many of the reads mapped to your reference genome. ... just to get an idea what could be the issue that caused the low number of reads. ( was a bit unclear from your blog)

Just for general information: meanwhile I had 3-4 attempts using the inline adapter approach from Lefrancois on hiSeq and also GAII. anyways, on the hiSeq I ended up with ~100 mio mapped reads out of ~200 clusters. However, this always required equimolar pools of 4 adapters to avoid possible problems due to biasing. I am still trying to figure out where the loss comes from.
... hope that is useful to anyone.

best, hel
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Old 12-10-2011, 06:59 AM   #49
ETHANol
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The low number of reads came from the leftover PCR oligos interfering with the binding of the library to the flow cell. This is fixed in the current version by purifying the final PCR with Ampure XP beads. Of course this is an issue known for years but what could I do, when it took Beckman-Culture months to figure out how fulfill an order to Greece.

I would still load a little more library then you would with a library made with Illumina's oligos as there seems to be a little magic something with the Illumina oligos. Now if Illumina would be nice enough to send me one of their TruSeq kits, I could figure this out better but again I've been waiting 2 months for a TruSeq RNA kit.

In my first run the percentages of mapped reads was good (something like 80%), the sequence quality was much better then the other samples on the same run (most likely due to the low cluster density). The cluster formed to successful cluster ratio was very high. These stats have stayed good in subsequent runs using the Ampure XP beads but with more reads.

My last library didn't go so well. It took 20 cycles total to amplify. Probably just not enough starting material, but annoying. It seems chromatin sheared with micrococcal libraries up more efficiently then chromatin sheared by sonication, but I have no side by side comparison here so it is speculative.
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Old 12-12-2011, 01:19 PM   #50
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Thanks,
regarding the leftover oligos - I re-run the libraries on a gel to get rid of them. Works perfect - actually better than with beads since my self annealed oligos are bigger than 100 (for whatever reason), and there is plenty of library material left to run 10 lanes.
I may have missed that in your protocol, but you are not using 5mC in your oligos right (might blow up the costs x10)?
Regarding the original illumina oligos, I've heard numerous problems in the ligation step using ChiP material, so people had to use ridiculous amounts of 50ng or above to get a decent library (which requires 4-5 pooled ChIPs) - I don't know if this is due to the methylation, the magic chemistry behind clustering, the size or a combination of all.

cheers, hel
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Old 08-04-2012, 10:08 AM   #51
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Hi everyone-
Has anyone figured out how little starting material one can use with Ethan's prep and still get enough library? Do you really have to use 10 ng starting material? I have seen protocols claiming to work down to 0.5 ng of starting material.

Thanks,
Joe
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Old 08-04-2012, 10:14 PM   #52
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We follow the protocol but with 5ng of ChIP DNA (Quantitation by Qubit), we did one at 2.5ng and it also worked. All with a total of 11 cycles of amplification; 4 pre-gel separation, 7 post gel excision) using Kapa HiFi.
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Old 08-05-2012, 12:55 AM   #53
ETHANol
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I've gone with as little as 2 ng and it worked out o.k. I can't remember the exact number of cycles though.
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Old 05-26-2013, 11:21 PM   #54
lihzhou
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Hi every one! It is my first post

I am very glad to see this topic. As our ChIP samples almost <5ng, the libraries prepared has very low concentration ~1ng/ul (Qubit HS) and always poor quality( high ratio of adapter dimer). Has anyone know the proper criterions for ChIP-seq libary?

Other request is that I failed to open the ETHANol protocol links http://ethanomics.files.wordpress.co...ip_truseq3.pdf or http://ethanomics.wordpress.com/. I'll feel so grateful if anyone can send the detail protocol to my email, [email protected] or [email protected]

Thanks,
lhz
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Old 05-26-2013, 11:32 PM   #55
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Quote:
Originally Posted by lihzhou View Post
Hi every one! It is my first post

I am very glad to see this topic. As our ChIP samples almost <5ng, the libraries prepared has very low concentration ~1ng/ul (Qubit HS) and always poor quality( high ratio of adapter dimer). Has anyone know the proper criterions for ChIP-seq libary?

Other request is that I failed to open the ETHANol protocol links http://ethanomics.files.wordpress.co...ip_truseq3.pdf or http://ethanomics.wordpress.com/. I'll feel so grateful if anyone can send the detail protocol to my email, [email protected] or [email protected]

Thanks,
lhz
Our version of Ethan's protocol can be downloaded from http://www.keatslab.org/protocols
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Old 05-26-2013, 11:39 PM   #56
ETHANol
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Wordpress.com has some problems. If you just keep clicking reload a bunch of times it will eventually load. Annoying and I'd like to move my blog somewhere more reliable but it never makes it up to the level of importance of something that actually gets done.

5 ng is doable. Sub 5 ng is find is usually pushing it.
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Old 05-26-2013, 11:55 PM   #57
lihzhou
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Hello, ETHANol and Jon_Keats

Thanks very much for replies!

I've succeded to download the protocol from http://www.keatslab.org/protocols and will try to prepare my libary again
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Old 04-24-2017, 08:33 PM   #58
warriert
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Default Minimum molar Requirement for ChIP-Seq libraries

Hey ETHANol,

This is my first post here and I'd first like to begin that your ChIP-Seq protocol at Ethanomics is religiously followed in my lab. :P

I have a slight conundrum however with some of the libraries I have prepared using this protocol of late. Since the ChIP samples I am working with are low in concentration due to the weak antibody, after 'successful' library preparation I am able to obtain a range of 2-10 nM of library sample for my ChIP samples.

I wish to know if this would be good enough to allow successful sequencing. The spread of the DNA ranges from 250 - 400 bp which normally denotes a decent library, however I am slightly worried about the concentrations though.

We will probably use a HiSeq 4000 for sequencing with 12 samples in a lane. In your expert opinion, do you think this concentration should enable decent sequencing for me?

warriert
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