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Old 07-23-2015, 10:54 AM   #1
DNA Sorcerer
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Default Nextera XT for prepping Chip-Seq sample?

Hello,
Illumina suggests using their TruSeq ChipSeq kit, but I wonder if the Nextera XT should work as well for chipseq sample (mainly because we already have an XT kit). This is for yeast and we plan on doing very short read sequencing (e.g. 50 cycles single). Any thoughts?
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Old 07-23-2015, 04:30 PM   #2
kerplunk412
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Because shearing is part of your ChIP process, you probably don't want to fragment your DNA any more. Also, with the XT kit the only fragments that will get adapters are those that have been cleaved, so you basically won't recover any full length molecules in your library. This will also, in theory, lower your coverage, as a single ChIPed molecule may be read multiple times, versus each read representing one molecule with a typical ligation-based library prep. That being said, ChIP samples often aren't as fragmented as the experimenter thinks they are, so additional fragmentation may help in some cases. But really, the ideal scenario is to get your fragments to the correct size during the ChIP process. It is always a good idea to check the size of your ChIPed material on a Bioanalyzer before starting library prep.

Also, for ligation-based library prep that there are many companies other than Illumina that provide less expensive kits that work as well if not better than the comparable Illumina kit, such as Bioo Scientific, NEB, and Kapa. As a disclaimer, I work for Bioo Scientific.
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Old 07-23-2015, 10:06 PM   #3
nucacidhunter
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Quote:
Originally Posted by DNA Sorcerer View Post
Hello,
Illumina suggests using their TruSeq ChipSeq kit, but I wonder if the Nextera XT should work as well for chipseq sample (mainly because we already have an XT kit). This is for yeast and we plan on doing very short read sequencing (e.g. 50 cycles single). Any thoughts?
It is possible, but you should ensure that pulled down DNA is clean and average fragment size is above 300 bp. Any chemical contaminant or proteins from pull down will have adverse effect on Nextera library prep.
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