SEQanswers

Go Back   SEQanswers > Applications Forums > Epigenetics



Similar Threads
Thread Thread Starter Forum Replies Last Post
ChIP-seq post-library prep qPCR ChipBrian Sample Prep / Library Generation 1 11-06-2014 07:47 AM
Best protocol for checking size of sonicated chIP fragments??? Narelle Sample Prep / Library Generation 0 05-22-2013 06:49 PM
Average fragment length post-sonication soleulloa Ion Torrent 3 11-16-2012 09:28 AM
advice on the size selection step in ChIP-seq protocol mboth Sample Prep / Library Generation 5 10-13-2011 02:59 AM
Yeast ChIP-seq; Sonication/Bioanalyzer question AaronS Sample Prep / Library Generation 1 01-14-2011 07:55 AM

Reply
 
Thread Tools
Old 08-15-2013, 10:14 AM   #1
omy567
Junior Member
 
Location: CA

Join Date: Aug 2013
Posts: 3
Default Post-ChIP Sonication Protocol

Hi guys. I'm working on a protocol to reduce the size of some post-ChIP DNA. I got nice sizing from 200-600 bp's in my sizing gel I ran before pulling down on my input material with my antibody of interest. However, after it seems that the epitope I am looking at is preferentially localizing to a larger fraction of chromatin (~1500 bp's). So, I'm trying to standardize a post-ChIP sonication protocol using a cup horn sonicator to get my size distribution down before I prepare some ChIP-seq libraries. Does anyone have or has heard a protocol using this sonicator that works to get a size around 250 bps? Thanks.
omy567 is offline   Reply With Quote
Old 08-19-2013, 08:18 AM   #2
NextGenSeq
Senior Member
 
Location: USA

Join Date: Apr 2009
Posts: 482
Default

We always shear our post-ChIP DNA again using a Covaris and their standard settings for the size we want. Usually we aim for 350 bp.

http://covarisinc.com/resources/protocols/
NextGenSeq is offline   Reply With Quote
Old 11-14-2013, 07:20 AM   #3
qsonica
Member
 
Location: New York

Join Date: Jun 2010
Posts: 11
Default

What model cup horn system do you have?
qsonica is offline   Reply With Quote
Old 12-05-2013, 02:27 PM   #4
Hamid
Senior Member
 
Location: San Diego, CA

Join Date: Sep 2009
Posts: 105
Default

chromatin shearing does generate a distribution, and the partially sheared material serve as better epitopes than the shears material. Hence you are you pulling down larger fragments that what you had expected. Depending on the energy used to shear the chromatin, your 200-600bp will likely have the epitope ripped off so they do not IP.
Consider reducing the energy/time of the shearing, and control the temperature of your samples to maintain epitope integrity.

Thank you

Hamid
Hamid is offline   Reply With Quote
Old 12-16-2013, 08:00 AM   #5
YanMar
Junior Member
 
Location: Sweden

Join Date: May 2013
Posts: 3
Default

Hi,

I'm experiencing the same problem for H3K4me3 and H3K27me3, despite that we shear the chromatin to 100-700 bp with the Covaris. The size shift is much more pronounced for H3K27me3 (aka I have a large peak >1000 bp).

We performed ChIP-qPCR on these ChIP samples as a QC before proceeding with the library preparation, and we see a satisfactory enrichment of correct histone target.

Now, I'm concerned about how this size-shift will affect the library preparation since the majority of the fragments are >1000 bp. Would it be possible to size select to remove all of those large fragments before library prep or perhaps "re-shear" the ChIP material again, before library prep?
YanMar is offline   Reply With Quote
Old 02-12-2014, 09:44 AM   #6
zwang0614
Member
 
Location: uk

Join Date: Feb 2014
Posts: 10
Default

Hi, YanMar.

I did not get what you said. When you said the size shift for H3K27me3, do you mean that the H3K27me3 can pull down the larger fragments >1000bp? In that case, if you remove the large fragments before library construction, how can you get the H3K27me3 pattern?

Wang
zwang0614 is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 01:17 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2019, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO