Hi guys,
This is my first post here so thanks all in advance for any comments. I have been working on ChIP for a while now and am now just making the jump to ChIP-sequencing. Anyway, I have just prepared my complete post-PCR TF library and have attached the Bioanalyzer results (High Sensitivity DNA chip). I have a pretty nice peak around the 250 bp area, which is great, but as you can see there is second peak around 150 (adapters probably) and also some lower peaks which to me look like primer dimers.
My question is simply, how do you guys think it looks and how should I proceed? My immediate thought was to re-do the size selection on a much longer gel and simply cut out a fresh band in the 250 bp range. I have enough library to get away with this but I have no idea how this will impact sequencing downstream. Is it out of the question to proceed as is? Of course the other option is to re-do the whole thing, which would mean me going all the way back to the cell culture stage!
Basic info:
ChIP - 10 million cells, using Magnify ChIP (Invitrogen)
Illumina prep - as per Illumina protocol except with a 1:30 adapter dilution. Size selection was done first on 2% gel (mini-gel), followed by PCR (18 cycles)
This is my first post here so thanks all in advance for any comments. I have been working on ChIP for a while now and am now just making the jump to ChIP-sequencing. Anyway, I have just prepared my complete post-PCR TF library and have attached the Bioanalyzer results (High Sensitivity DNA chip). I have a pretty nice peak around the 250 bp area, which is great, but as you can see there is second peak around 150 (adapters probably) and also some lower peaks which to me look like primer dimers.
My question is simply, how do you guys think it looks and how should I proceed? My immediate thought was to re-do the size selection on a much longer gel and simply cut out a fresh band in the 250 bp range. I have enough library to get away with this but I have no idea how this will impact sequencing downstream. Is it out of the question to proceed as is? Of course the other option is to re-do the whole thing, which would mean me going all the way back to the cell culture stage!
Basic info:
ChIP - 10 million cells, using Magnify ChIP (Invitrogen)
Illumina prep - as per Illumina protocol except with a 1:30 adapter dilution. Size selection was done first on 2% gel (mini-gel), followed by PCR (18 cycles)
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