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ChIP-Seq Library Prep: PCR bubble? No enrichment over input
Hello everyone,
I have used NEBnext® Ultra™ II DNA library prep kit for Illumina to make
ChIP-Seq libraries with 1.2 ng DNA (quantified by Qubit). I did the PCR for
12 cycles, as the recommended number of cycles for 1 ng was 12-13 cycles. When I ran my libraries on a bioanalyzer, I noticed that although the input samples showed one peak at the expected size, the pull-down libraries showed a larger second peak, whether it was for the IgG or the protein-of-interest. This is as shown on the left hand side of this photo: https://drive.google.com/file/d/1JYE...ew?usp=sharing
The sequencing facility said that it would be ok to proceed with sequencing, so I diluted all my libraries to the required concentration (0.6 ng/ul) and pooled them in equal volumes. Prior to pooling, I took an aliquot and did a qPCR to further confirm that my pull-down worked, using primers against my region of interest and I did get an enrichment of the pull down over the input.
However, when I got the sequencing results back and the bioinformatics analysis was done, I lost this enrichment at the promoter that I validated by qPCR. The link here might help clarify what I mean more: https://drive.google.com/file/d/1tYe...ew?usp=sharing
Basically, I can see that my input and pull down sample are both enriched at the promoter, however, the input seems to have much more reads, to the extent that when the pull down is normalized over the input, I don't see an enrichment anymore.
I thought the problem could be a PCR bubble and hence did one cycle of reconditioning PCR, whereby I diluted the libraries 1:5 and added the Q5 Master Mix and primers in excess and ran this through 1 PCR cycle. However, I got the same profile as described in the first link I shared / this link on the right hand side: https://drive.google.com/file/d/1JYE...ew?usp=sharing
I am not quite sure where things could have gone wrong..
It is not a fragmentation issue because the input sample (1st lane) is properly fragmented and it is from the same lysate from which the pull-downs were done.
The Qubit concentrations of the input library is quite low compared to the pull-downs, even though I start the library prep with the same quantities. As a result, I end up taking a larger volume of the input samples - unless this means that something is wrong with the Qubit measurements?
Any suggestions on what I can do would be greatly appreciated.
I have used NEBnext® Ultra™ II DNA library prep kit for Illumina to make
ChIP-Seq libraries with 1.2 ng DNA (quantified by Qubit). I did the PCR for
12 cycles, as the recommended number of cycles for 1 ng was 12-13 cycles. When I ran my libraries on a bioanalyzer, I noticed that although the input samples showed one peak at the expected size, the pull-down libraries showed a larger second peak, whether it was for the IgG or the protein-of-interest. This is as shown on the left hand side of this photo: https://drive.google.com/file/d/1JYE...ew?usp=sharing
The sequencing facility said that it would be ok to proceed with sequencing, so I diluted all my libraries to the required concentration (0.6 ng/ul) and pooled them in equal volumes. Prior to pooling, I took an aliquot and did a qPCR to further confirm that my pull-down worked, using primers against my region of interest and I did get an enrichment of the pull down over the input.
However, when I got the sequencing results back and the bioinformatics analysis was done, I lost this enrichment at the promoter that I validated by qPCR. The link here might help clarify what I mean more: https://drive.google.com/file/d/1tYe...ew?usp=sharing
Basically, I can see that my input and pull down sample are both enriched at the promoter, however, the input seems to have much more reads, to the extent that when the pull down is normalized over the input, I don't see an enrichment anymore.
I thought the problem could be a PCR bubble and hence did one cycle of reconditioning PCR, whereby I diluted the libraries 1:5 and added the Q5 Master Mix and primers in excess and ran this through 1 PCR cycle. However, I got the same profile as described in the first link I shared / this link on the right hand side: https://drive.google.com/file/d/1JYE...ew?usp=sharing
I am not quite sure where things could have gone wrong..
It is not a fragmentation issue because the input sample (1st lane) is properly fragmented and it is from the same lysate from which the pull-downs were done.
The Qubit concentrations of the input library is quite low compared to the pull-downs, even though I start the library prep with the same quantities. As a result, I end up taking a larger volume of the input samples - unless this means that something is wrong with the Qubit measurements?
Any suggestions on what I can do would be greatly appreciated.