Hello all,
I've recently found this forum and wanted to ask for feedback about the quality of the attached ChIP-seq libraries.
The relevant information is:
Used Bioo Scientific NextFlex Barcode Adapters with a concentration of 0.6uM for a total amount of 10ng of DNA.
I size select the library to remove excess adapters and select a size range of templates around the 300bp size using a E-Gel Size select system
Following the size selection I did PCR amplification for 15 cycles of half of the sample.
Afterwards I did an Ampure bead purification to further clean up the sample of fragments with the wrong size.
However in most of my samples I still obtained adapter dimers peaks. Since adapter dimers are undesirable since they compete with the actual sample when sequencing in the Illumina Genome Analyzer that we are going to use I wanted to know if the amount of adapter dimers found in these samples is significant and I should redo the libraries maybe with a smaller amount of adapters.
I am also having issues with correctly determining the adequate amount of DNA in my samples: What I consistently observe is that input samples always have more DNA than IP samples in the E-Gel Recovery System even when I am always using 10ng of sample. So it's impossible to adjust the amount of DNA beforehand, if I am seeing variations only after the adapter ligation step.
So is any of those samples usable? I would like to know specifically if sample 2 can be used since I have a very limited amount of DNA of that material.
Thanks for the feedback.
I've recently found this forum and wanted to ask for feedback about the quality of the attached ChIP-seq libraries.
The relevant information is:
Used Bioo Scientific NextFlex Barcode Adapters with a concentration of 0.6uM for a total amount of 10ng of DNA.
I size select the library to remove excess adapters and select a size range of templates around the 300bp size using a E-Gel Size select system
Following the size selection I did PCR amplification for 15 cycles of half of the sample.
Afterwards I did an Ampure bead purification to further clean up the sample of fragments with the wrong size.
However in most of my samples I still obtained adapter dimers peaks. Since adapter dimers are undesirable since they compete with the actual sample when sequencing in the Illumina Genome Analyzer that we are going to use I wanted to know if the amount of adapter dimers found in these samples is significant and I should redo the libraries maybe with a smaller amount of adapters.
I am also having issues with correctly determining the adequate amount of DNA in my samples: What I consistently observe is that input samples always have more DNA than IP samples in the E-Gel Recovery System even when I am always using 10ng of sample. So it's impossible to adjust the amount of DNA beforehand, if I am seeing variations only after the adapter ligation step.
So is any of those samples usable? I would like to know specifically if sample 2 can be used since I have a very limited amount of DNA of that material.
Thanks for the feedback.
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