ChIP-seq DNA concentration

jazz · 10-05-2011, 10:34 AM

Hi everyone,
I am doing a ChIP-seq using Illumina platform. After IP, I have less than 10 ng of DNA, while I have at least 10-fold excess in Input. My question is whether it is important to use same amount of Input and ChIP DNA before library preparation. The reason I am worried about this is 2-fold
a) If using different IP and input DNA before library prep, how else is the data normalized?
b) If starting with different amounts of DNA in the final PCR amplification step, there could be a differential amplification of some regions, skewing the data.

What are your thoughts.
Thanks for all inputs.

kthai · 10-07-2011, 08:16 AM

Yes, I completely agree with you. When I prepare my ChIP-Seq libraries, the input and IP concentrations are the same to reduce any biases, especially during the amplification stage.

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10-05-2011, 10:34 AM	#1
jazz Member Location: us Join Date: Nov 2008 Posts: 28	ChIP-seq DNA concentration Hi everyone, I am doing a ChIP-seq using Illumina platform. After IP, I have less than 10 ng of DNA, while I have at least 10-fold excess in Input. My question is whether it is important to use same amount of Input and ChIP DNA before library preparation. The reason I am worried about this is 2-fold a) If using different IP and input DNA before library prep, how else is the data normalized? b) If starting with different amounts of DNA in the final PCR amplification step, there could be a differential amplification of some regions, skewing the data. What are your thoughts. Thanks for all inputs.

10-07-2011, 08:16 AM	#2
kthai Junior Member Location: Boston Join Date: Jul 2010 Posts: 6	Yes, I completely agree with you. When I prepare my ChIP-Seq libraries, the input and IP concentrations are the same to reduce any biases, especially during the amplification stage.