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Old 05-13-2009, 03:48 AM   #1
spudybudy
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Location: London

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Default Bioanalyser traces + QC postPCR. ChIPseq Illumina protocol

Hi everyone.
I have been trying to get ChIPseq libraries. I started with 10ng as suggested by Illumina and got a Bioanalyser trace. Problems: the trace is actually two quite-fused peaks. The first at around 165bp and the second at around 224bp (I cut the gel from 125-150bp to 300bp).
Is this normal?
Another question would be if anyone does a QC after the protocol's PCR. imagine you start with the INPUT sample (pre-immunoprecipitated DNA. Your ChIP background control, basically) which is normal fragmented genomic DNA. Before library preparation, you have the same amount for all genomic locations (as seen on RT-PCR). But after library PCR, there is a quite strong bias for some regions compared to others. Is this normal? do you do this QC step or go direcly into sequencing if the library looks fine on the Bioanalyser?
Cheers everyone in advance!
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Old 08-26-2009, 05:14 AM   #2
Susanne
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Did you try to run the IP-ed material (on the Bioanalyzer) before sample prep.? This may give you a clue if the size distribution looked like this before adding the adapters.
Sorry, not the complete solution, but maybe it give a hint...
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