ChipSeq library construction from tissue and DNA purity issues

Theorbe27 · 02-18-2011, 02:18 PM

Hi,

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

MK

edawad · 02-23-2011, 05:51 PM

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.

kalidaemon · 06-10-2011, 06:59 AM

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.

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02-18-2011, 02:18 PM	#1
Theorbe27 Junior Member Location: Boston Join Date: Feb 2011 Posts: 3	ChipSeq library construction from tissue and DNA purity issues Hi, 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 MK

02-23-2011, 05:51 PM	#2
edawad Member 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.

06-10-2011, 06:59 AM	#3
kalidaemon Member 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.