Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation

Similar Threads
Thread Thread Starter Forum Replies Last Post
Sequencing Library Construction of Solexa chloe_giselle Illumina/Solexa 2 12-11-2011 11:56 AM
Constructing library of tissue-specific genes johannes.helmuth RNA Sequencing 0 10-20-2011 02:20 AM
miRNA library construction/sequencing lvcosme General 0 09-20-2011 01:30 PM
ChIPseq on tissue - eliminating background Theorbe27 Epigenetics 4 06-23-2011 08:53 AM
Ti vs. FLX library construction HMorrison 454 Pyrosequencing 1 10-29-2009 05:51 AM

Thread Tools
Old 02-18-2011, 01:18 PM   #1
Junior Member
Location: Boston

Join Date: Feb 2011
Posts: 3
Question ChipSeq library construction from tissue and DNA purity issues


we are currently trying to construct a ChIP-Seq library from a tissue sample. To complicate things, the transcription factor we would like to assay for is only expressed in a fraction (maybe 3% or so) of the cells present in the sample.
I am able to get about a 5fold enrichment for a candidate gene by ChIP-qPCR.
However, I am concerned about going forward with library construction because of the signal/noise ratio I may have to expect. Furthermore, my 260/280 ratios of the chipped DNAs as by Nanodrop suck (~ 1.3 after ProK digest and 2x PCI cleanup).
Has anybody ever done something similar?
Has anybody used Qiagen columns to clean up chipped DNA and found these to be OK even for small amounts of DNA? Would anybody perhaps have some other advice how to further purify the DNA?

Thanks for your help

Theorbe27 is offline   Reply With Quote
Old 02-23-2011, 04:51 PM   #2
Location: bay area

Join Date: Mar 2010
Posts: 10

Don't worry about nanodropping your DNA after ChIP. The concentration is most likely <10ng/ul given your conditions so if anything you should be using a Qubit. Just proceed with your favorite chip-seq library prep, using qiagen minelute columns (you can try double-eluting with heated EB, e.g. for a 20 ul elution elute twice with 10 ul hot EB). Do a few test amplifications (15, 18, 21 cycles) and try to re-validate with qPCR on the final library. If that passes and you have sufficient DNA to put on a flowcell, you should be clear.
edawad is offline   Reply With Quote
Old 06-10-2011, 05:59 AM   #3
Location: Boston

Join Date: Sep 2010
Posts: 14

I'm doing the same thing and have been able to increase my yield using phenol/chloroform extraction with phase-lock gels to clean up my ChIPped DNA after reverse cross-linking. Also, you might want to try quantitating your DNA with a flourescence-based ds DNA kit (I like Invitrogen Quantit-IT) because your sample concentration might be below the detection limit of the nanodrop.
kalidaemon is offline   Reply With Quote

chip seq, contamination, noise, tissue

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 09:00 PM.

Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2021, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO