Tweet
Hi,
I just ran our first ChIP-seq experiments for 4 histone marks, 1 input, and three TFs. While the histone marks all worked very well, the TFs worked poorly with very low signal to noise and high rates of duplication (~40%).
I did a lot of optimization for the shearing (300-400 bp avg.) and IP, and was obtaining >10 to 60 fold enrichment for the TFs by qPCR. So I took the ChIP DNA forward, and had >10 ng for the TFs (pooling 4 ChIPs together for each) and 20 ng for the histones starting out for the library prep. After the 18 cycles of PCR and gel extraction size-selection (250-600 bp), I noticed that there were a lot of these "bubble products" in the TF libraries (didn't know what they were at the time). So I re-size-selected by Pippen Prep (250-600 bp) to see if I could get rid of them, but the high MW shoulder remained by bioanalyzer. I re-size-selected all samples to be consistent.
Anyways, we decided to quant. the libraries by KAPA and went for sequencing. As I mentioned earlier, the histones worked well, but the TFs had high rates of duplication and bad signal to noise (aka they looked like crap). I suspected the library prep since there were a lot of PCR duplicates, but I'm confused since the histones went through the same library prep (processed at the same time), and they turned out OK despite roughly a similar amount of input DNA (10, 16, and 19 ng vs. 20 for all of the histones).
I ran qPCR on the ChIP libraries (I now know to do this before sequencing...), and noticed that the profile for the enrichment changed drastically after the library prep. So I'm going through the library prep process again to figure out what happened, but was wondering if anyone had any insights to where things went awry given that the histone preps worked...
Another possibility is that the IP was crap despite what looks like >10 to 60-fold enrichment by PCR on my ChIPs before library prep, but seems like there are a lot of things pointing to the library prep being bad (i.e., the high rate of PCR duplicates and the bubble products).
Thanks ahead of time for the help!
Brian
I just ran our first ChIP-seq experiments for 4 histone marks, 1 input, and three TFs. While the histone marks all worked very well, the TFs worked poorly with very low signal to noise and high rates of duplication (~40%).
I did a lot of optimization for the shearing (300-400 bp avg.) and IP, and was obtaining >10 to 60 fold enrichment for the TFs by qPCR. So I took the ChIP DNA forward, and had >10 ng for the TFs (pooling 4 ChIPs together for each) and 20 ng for the histones starting out for the library prep. After the 18 cycles of PCR and gel extraction size-selection (250-600 bp), I noticed that there were a lot of these "bubble products" in the TF libraries (didn't know what they were at the time). So I re-size-selected by Pippen Prep (250-600 bp) to see if I could get rid of them, but the high MW shoulder remained by bioanalyzer. I re-size-selected all samples to be consistent.
Anyways, we decided to quant. the libraries by KAPA and went for sequencing. As I mentioned earlier, the histones worked well, but the TFs had high rates of duplication and bad signal to noise (aka they looked like crap). I suspected the library prep since there were a lot of PCR duplicates, but I'm confused since the histones went through the same library prep (processed at the same time), and they turned out OK despite roughly a similar amount of input DNA (10, 16, and 19 ng vs. 20 for all of the histones).
I ran qPCR on the ChIP libraries (I now know to do this before sequencing...), and noticed that the profile for the enrichment changed drastically after the library prep. So I'm going through the library prep process again to figure out what happened, but was wondering if anyone had any insights to where things went awry given that the histone preps worked...
Another possibility is that the IP was crap despite what looks like >10 to 60-fold enrichment by PCR on my ChIPs before library prep, but seems like there are a lot of things pointing to the library prep being bad (i.e., the high rate of PCR duplicates and the bubble products).
Thanks ahead of time for the help!
Brian
Comment