SEQanswers

Go Back   SEQanswers > Applications Forums > Epigenetics



Similar Threads
Thread Thread Starter Forum Replies Last Post
ChIP-Seq: Single-tube linear DNA amplification (LinDA) for robust ChIP-seq. Newsbot! Literature Watch 19 02-29-2016 07:08 PM
Whole-genome amplification prior to Illumina sequencing? Crawford505 Illumina/Solexa 24 03-20-2012 01:40 PM
Sequencing WGA ChIP DNA fedora Illumina/Solexa 5 01-20-2011 03:17 AM
Whole genome amplification prior to ChIP-Seq aperera Sample Prep / Library Generation 1 10-08-2010 10:51 AM
Quality testing of ChIP-seq library prior to sequencing. The Bric Sample Prep / Library Generation 8 09-17-2010 04:10 PM

Reply
 
Thread Tools
Old 10-08-2010, 10:41 AM   #1
angelawu
Member
 
Location: Stanford, CA

Join Date: Feb 2010
Posts: 12
Default WGA Amplification for ChIP samples prior to Seq?

Hi,

I am working with small cell number ChIP-seq, and was wondering if anyone has any experience doing a WGA or similar after the ChIP step, before going on to make the library and seq?

I have tried doing the Illumina PCR amplification protocol after attaching the adapters, but it results in a lot of bias based on size and GC content, which for small samples basically kills the specificity of the experiment.

I'm considering the WGA kit from Sigma... any experience/guesses on whether that might work better? I'm also open to other kits people have used.

Thanks in advance!
angelawu is offline   Reply With Quote
Old 10-08-2010, 11:08 AM   #2
aperera
Member
 
Location: Kansas City

Join Date: Jan 2008
Posts: 23
Default

Can you tell me what sort of bias towards GC content you are seeing?
aperera is offline   Reply With Quote
Old 10-08-2010, 11:46 AM   #3
angelawu
Member
 
Location: Stanford, CA

Join Date: Feb 2010
Posts: 12
Default

Basically the GC rich sequences will be over-represented. It has been published here:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2862719/
angelawu is offline   Reply With Quote
Old 10-08-2010, 02:14 PM   #4
Chipper
Senior Member
 
Location: Sweden

Join Date: Mar 2008
Posts: 324
Default

Don't think they used wga but you may want to check this protocol:
http://www.nature.com/nmeth/journal/...meth.1478.html
Chipper is offline   Reply With Quote
Old 10-22-2010, 07:42 AM   #5
JHU-ChIPmaniac
Member
 
Location: Baltimore

Join Date: Jul 2010
Posts: 21
Default

I don't have any experience with small cell numbers. Are you pulling down a histone modification or a TF? The only publications I have seen using low cell numbers (10,000 cells) and successful ChIP-seq were using antibodies against histones. Has anyone sequenced a ChIP-seq library from a TF and low cell numbers?
JHU-ChIPmaniac is offline   Reply With Quote
Old 04-12-2012, 06:30 AM   #6
linampli
Junior Member
 
Location: france

Join Date: Mar 2012
Posts: 7
Default

check this out...
Single-tube linear DNA amplification for genome-wide studies using a few thousand cells.
Shankaranarayanan P, Mendoza-Parra MA, van Gool W, Trindade LM, Gronemeyer H.
Nat Protoc. 2012 Jan 26;7(2):328-38. doi: 10.1038/nprot.2011.447.
Nat Methods. 2011 Jun 5;
linampli is offline   Reply With Quote
Reply

Tags
amplification, chip-seq, low cell number

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 04:20 PM.


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