SEQanswers

Go Back   SEQanswers > Literature Watch



Similar Threads
Thread Thread Starter Forum Replies Last Post
ChIP-Seq: CistromeMap: A knowledgebase and web server for ChIP-Seq and DNase-Seq stud Newsbot! Literature Watch 0 04-13-2012 03:30 AM
ChIP-Seq: ChIP-Array: combinatory analysis of ChIP-seq/chip and microarray gene expre Newsbot! Literature Watch 0 05-19-2011 03:50 AM
ChIP-Seq: ChIP-chip versus ChIP-seq: Lessons for experimental design and data analysi Newsbot! Literature Watch 0 03-02-2011 03:50 AM
ChIP-seq in cancer cell lines captainentropy General 2 09-14-2010 02:42 PM
ChIP-Seq: ChIPpeakAnno: a Bioconductor package to annotate ChIP-seq and ChIP-chip dat Newsbot! Literature Watch 0 05-13-2010 03:00 AM

Reply
 
Thread Tools
Old 11-23-2012, 03:00 AM   #1
Newsbot!
RSS Posting Maniac
 

Join Date: Feb 2008
Posts: 1,439
Default ChIP-Seq: Limitations and possibilities of low cell number ChIP-seq.

Syndicated from PubMed RSS Feeds

Limitations and possibilities of low cell number ChIP-seq.

BMC Genomics. 2012 Nov 21;13(1):645

Authors: Gilfillan GD, Hughes T, Sheng Y, Hjorthaug HS, Straub T, Gervin K, Harris JR, Undlien DE, Lyle R

Abstract
ABSTRACT: BACKGROUND: Chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq) offers high resolution, genome-wide analysis of DNA-protein interactions. However, current standard methods require abundant starting material in the range of 1--20 million cells per immunoprecipitation, and remain a bottleneck to the acquisition of biologically relevant epigenetic data. Using a ChIP-seq protocol optimised for low cell numbers (down to 100,000 cells / IP), we examined the performance of the ChIP-seq technique on a series of decreasing cell numbers. RESULTS: We present an enhanced native ChIP-seq method tailored to low cell numbers that represents a 200-fold reduction in input requirements over existing protocols. The protocol was tested over a range of starting cell numbers covering three orders of magnitude, enabling determination of the lower limit of the technique. At low input cell numbers, increased levels of unmapped and duplicate reads reduce the number of unique reads generated, and can drive up sequencing costs and affect sensitivity if ChIP is attempted from too few cells. CONCLUSIONS: The optimised method presented here considerably reduces the input requirements for performing native ChIP-seq. It extends the applicability of the technique to isolated primary cells and rare cell populations (e.g. biobank samples, stem cells), and in many cases will alleviate the need for cell culture and any associated alteration of epigenetic marks. However, this study highlights a challenge inherent to ChIP-seq from low cell numbers: as cell input numbers fall, levels of unmapped sequence reads and PCR-generated duplicate reads rise. We discuss a number of solutions to overcome the effects of reducing cell number that may aid further improvements to ChIP performance.


PMID: 23171294 [PubMed - as supplied by publisher]



More...
Newsbot! 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 11:36 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2018, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO